Granulocyte colony stimulating factor: remodeling and glycoconjugation of G-CSF

ABSTRACT

The invention includes methods and compositions for remodeling a peptide molecule, including the addition or deletion of one or more glycosyl groups to a peptide, and/or the addition of a modifying group to a peptide.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of prior ApplicationNo. PCT/US02/32263, filed Oct. 9, 2002; Provisional Patent ApplicationNo. 60/448,381, filed Feb. 19, 2003 (converted to non-provisionalapplication, same filing date, serial number not yet assigned);Provisional Patent Application No. 60/438,582, filed Jan. 6, 2003(converted to non-provisional application, same filing date, serialnumber not yet assigned); Provisional Patent Application No. 60/407,527,filed Aug. 28, 2002; Provisional Patent Application No. 60/404,249,filed Aug. 16, 2002; Provisional Patent Application No. 60/396,594,filed Jul. 17, 2002; Provisional Patent Application No. 60/391,777,filed Jun. 25, 2002; Provisional Patent Application No. 60/387,292,filed Jun. 7, 2002; Provisional Patent Application No. 60/334,301, filedNov. 28, 2001; Provisional Patent Application No. 60/334,233, filed Nov.28, 2001; Provisional Patent Application No. 60/344,692, filed Oct. 19,2001; and Provisional Patent Application No. 60/328,523, filed Oct. 10,2001.

BACKGROUND OF THE INVENTION

[0002] Most naturally occurring peptides contain carbohydrate moietiesattached to the peptide via specific linkages to a select number ofamino acids along the length of the primary peptide chain. Thus, manynaturally occurring peptides are termed “glycopeptides.” The variabilityof the glycosylation pattern on any given peptide has enormousimplications for the function of that peptide. For example, thestructure of the N-linked glycans on a peptide can impact variouscharacteristics of the peptide, including the protease susceptibility,intracellular trafficking, secretion, tissue targeting, biologicalhalf-life and antigenicity of the peptide in a cell or organism. Thealteration of one or more of these characteristics greatly affects theefficacy of a peptide in its natural setting, and also affects theefficacy of the peptide as a therapeutic agent in situations where thepeptide has been generated for that purpose.

[0003] The carbohydrate structure attached to the peptide chain is knownas a “glycan” molecule. The specific glycan structure present on apeptide affects the solubility and aggregation characteristics of thepeptide, the folding of the primary peptide chain and therefore itsfunctional or enzymatic activity, the resistance of the peptide toproteolytic attack and the control of proteolysis leading to theconversion of inactive forms of the peptide to active forms.Importantly, terminal sialic acid residues present on the glycanmolecule affect the length of the half life of the peptide in themammalian circulatory system. Peptides whose glycans do not containterminal sialic acid residues are rapidly removed from the circulationby the liver, an event which negates any potential therapeutic benefitof the peptide.

[0004] The glycan structures found in naturally occurring glycopeptidesare typically divided into two classes, N-linked and O-linked glycans.

[0005] Peptides expressed in eukaryotic cells are typicallyN-glycosylated on asparagine residues at sites in the peptide primarystructure containing the sequence asparagine-X-serine/threonine where Xcan be any amino acid except proline and aspartic acid. The carbohydrateportion of such peptides is known as an N-linked glycan. The earlyevents of N-glycosylation occur in the endoplasmic reticulum (ER) andare identical in mammals, plants, insects and other higher eukaryotes.First, an oligosaccharide chain comprising fourteen sugar residues isconstructed on a lipid carrier molecule. As the nascent peptide istranslated and translocated into the ER, the entire oligosaccharidechain is transferred to the amide group of the asparagine residue in areaction catalyzed by a membrane bound glycosyltransferase enzyme. TheN-linked glycan is further processed both in the ER and in the Golgiapparatus. The further processing generally entails removal of some ofthe sugar residues and addition of other sugar residues in reactionscatalyzed by glycosidases and glycosyltransferases specific for thesugar residues removed and added.

[0006] Typically, the final structures of the N-linked glycans aredependent upon the organism in which the peptide is produced. Forexample, in general, peptides produced in bacteria are completelyunglycosylated. Peptides expressed in insect cells contain high mannoseand paunci-mannose N-linked oligosaccharide chains, among others.Peptides produced in mammalian cell culture are usually glycosylateddifferently depending, e.g., upon the species and cell cultureconditions. Even in the same species and under the same conditions, acertain amount of heterogeneity in the glycosyl chains is sometimesencountered. Further, peptides produced in plant cells comprise glycanstructures that differ significantly from those produced in animalcells. The dilemma in the art of the production of recombinant peptides,particularly when the peptides are to be used as therapeutic agents, isto be able to generate peptides that are correctly glycosylated, i.e.,to be able to generate a peptide having a glycan structure thatresembles, or is identical to that present on the naturally occurringform of the peptide. Most peptides produced by recombinant meanscomprise glycan structures that are different from the naturallyoccurring glycans.

[0007] A variety of methods have been proposed in the art to customizethe glycosylation pattern of a peptide including those described in WO99/22764, WO 98/58964, WO 99/54342 and U.S. Pat. No. 5,047,335, amongothers. Essentially, many of the enzymes required for the in vitroglycosylation of peptides have been cloned and sequenced. In someinstances, these enzymes have been used in vitro to add specific sugarsto an incomplete glycan molecule on a peptide. In other instances, cellshave been genetically engineered to express a combination of enzymes anddesired peptides such that addition of a desired sugar moiety to anexpressed peptide occurs within the cell.

[0008] Peptides may also be modified by addition of O-linked glycans,also called mucin-type glycans because of their prevalence on mucinousglycopeptide. Unlike N-glycans that are linked to asparagine residuesand are formed by en bloc transfer of oligosaccharide from lipid-boundintermediates, O-glycans are linked primarily to serine and threonineresidues and are formed by the stepwise addition of sugars fromnucleotide sugars (Tanner et al., Biochim. Biophys. Acta. 906:81-91(1987); and Hounsell et al., Glycoconj. J. 13:19-26 (1996)). Peptidefunction can be affected by the structure of the O-linked glycanspresent thereon. For example, the activity of P-selectin ligand isaffected by the O-linked glycan structure present thereon. For a reviewof O-linked glycan structures, see Schachter and Brockhausen, TheBiosynthesis of Branched O-Linked Glycans, 1989, Society forExperimental Biology, pp. 1-26 (Great Britain). Other glycosylationpatterns are formed by linking glycosylphosphatidylinositol to thecarboxyl-terminal carboxyl group of the protein (Takeda et al., TrendsBiochem. Sci. 20:367-371 (1995); and Udenfriend et al., Ann. Rev.Biochem. 64:593-591 (1995).

[0009] Although various techniques currently exist to modify theN-linked glycans of peptides, there exists in the art the need for agenerally applicable method of producing peptides having a desired,i.e., a customized glycosylation pattern. There is a particular need inthe art for the customized in vitro glycosylation of peptides, where theresulting peptide can be produced at industrial scale. This and otherneeds are met by the present invention.

[0010] The administration of glycosylated and non-glycosylated peptidesfor engendering a particular physiological response is well known in themedicinal arts. Among the best known peptides utilized for this purposeis insulin, which is used to treat diabetes. Enzymes have also been usedfor their therapeutic benefits. A major factor, which has limited theuse of therapeutic peptides is the immunogenic nature of most peptides.In a patient, an immunogenic response to an administered peptide canneutralize the peptide and/or lead to the development of an allergicresponse in the patient. Other deficiencies of therapeutic peptidesinclude suboptimal potency and rapid clearance rates. The problemsinherent in peptide therapeutics are recognized in the art, and variousmethods of eliminating the problems have been investigated. To providesoluble peptide therapeutics, synthetic polymers have been attached tothe peptide backbone.

[0011] Poly(ethylene glycol) (“PEG”) is an exemplary polymer that hasbeen conjugated to peptides. The use of PEG to derivatize peptidetherapeutics has been demonstrated to reduce the immunogenicity of thepeptides and prolong the clearance time from the circulation. Forexample, U.S. Pat. No. 4,179,337 (Davis et al.) concerns non-immunogenicpeptides, such as enzymes and peptide hormones coupled to polyethyleneglycol (PEG) or polypropylene glycol. Between 10 and 100 moles ofpolymer are used per mole peptide and at least 15% of the physiologicalactivity is maintained.

[0012] WO 93/15189 (Veronese et al.) concerns a method to maintain theactivity of polyethylene glycol-modified proteolytic enzymes by linkingthe proteolytic enzyme to a macromolecularized inhibitor. The conjugatesare intended for medical applications.

[0013] The principal mode of attachment of PEG, and its derivatives, topeptides is a non-specific bonding through a peptide amino acid residue.For example, U.S. Pat. No. 4,088,538 discloses an enzymatically activepolymer-enzyme conjugate of an enzyme covalently linked to PEG.Similarly, U.S. Pat. No. 4,496,689 discloses a covalently attachedcomplex of α-1 protease inhibitor with a polymer such as PEG ormethoxypoly(ethylene glycol) (“mPEG”). Abuchowski et al. (J. Biol. Chem.252: 3578 (1977) discloses the covalent attachment of MPEG to an aminegroup of bovine serum albumin. U.S. Pat. No. 4,414,147 discloses amethod of rendering interferon less hydrophobic by conjugating it to ananhydride of a dicarboxylic acid, such as poly(ethylene succinicanhydride). PCT WO 87/00056 discloses conjugation of PEG andpoly(oxyethylated) polyols to such proteins as interferon-β,interleukin-2 and immunotoxins. EP 154,316 discloses and claimschemically modified lymphokines, such as IL-2 containing PEG bondeddirectly to at least one primary amino group of the lymphokine. U.S.Pat. No. 4,055,635 discloses pharmaceutical compositions of awater-soluble complex of a proteolytic enzyme linked covalently to apolymeric substance such as a polysaccharide.

[0014] Another mode of attaching PEG to peptides is through thenon-specific oxidation of glycosyl residues on a peptide. The oxidizedsugar is utilized as a locus for attaching a PEG moiety to the peptide.For example, M'Timkulu (WO 94/05332) discloses the use of a hydrazine-or amino-PEG to add PEG to a glycoprotein. The glycosyl moieties arerandomly oxidized to the corresponding aldehydes, which are subsequentlycoupled to the amino-PEG. See also, Bona et al. (WO 96/40731), where aPEG is added to an immunoglobulin molecule by enzymatically oxidizing aglycan on the immunoglobulin and then contacting the glycan with anamino-PEG molecule.

[0015] In each of the methods described above, poly(ethylene glycol) isadded in a random, non-specific manner to reactive residues on a peptidebackbone. For the production of therapeutic peptides, it is clearlydesirable to utilize a derivatization strategy that results in theformation of a specifically labeled, readily characterizable,essentially homogeneous product.

[0016] Two principal classes of enzymes are used in the synthesis ofcarbohydrates, glycosyltransferases (e.g., sialyltransferases,oligosaccharyltransferases, N-acetylglucosaminyltransferases), andglycosidases. The glycosidases are further classified as exoglycosidases(e.g., β-mannosidase, β-glucosidase), and endoglycosidases (e.g.,Endo-A, Endo-M). Each of these classes of enzymes has been successfullyused synthetically to prepare carbohydrates. For a general review, see,Crout et al., Curr. Opin. Chem. Biol. 2: 98-111 (1998).

[0017] Glycosyltransferases modify the oligosaccharide structures onpeptides. Glycosyltransferases are effective for producing specificproducts with good stereochemical and regiochemical control.Glycosyltransferases have been used to prepare oligosaccharides and tomodify terminal N- and O-linked carbohydrate structures, particularly onpeptides produced in mammalian cells. For example, the terminaloligosaccharides of glycopeptides have been completely sialylated and/orfucosylated to provide more consistent sugar structures, which improvesglycopeptide pharmacodynamics and a variety of other biologicalproperties. For example, β-1,4-galactosyltransferase is used tosynthesize lactosamine, an illustration of the utility ofglycosyltransferases in the synthesis of carbohydrates (see, e.g., Wonget al., J. Org. Chem. 47: 5416-5418 (1982)). Moreover, numeroussynthetic procedures have made use of α-sialyltransferases to transfersialic acid from cytidine-5′-monophospho-N-acetylneuraminic acid to the3-OH or 6-OH of galactose (see, e.g., Kevin et al., Chem. Eur. J. 2:1359-1362 (1996)). Fucosyltransferases are used in synthetic pathways totransfer a fucose unit from guanosine-5′-diphosphofucose to a specifichydroxyl of a saccharide acceptor. For example, Ichikawa prepared sialylLewis-X by a method that involves the fucosylation of sialylatedlactosamine with a cloned fucosyltransferase (Ichikawa et al., J. Am.Chem. Soc. 114: 9283-9298 (1992)). For a discussion of recent advancesin glycoconjugate synthesis for therapeutic use see, Koeller et al.,Nature Biotechnology 18: 835-841 (2000). See also, U.S. Pat. Nos.5,876,980; 6,030,815; 5,728,554; 5,922,577; and WO/9831826.

[0018] Glycosidases can also be used to prepare saccharides.Glycosidases normally catalyze the hydrolysis of a glycosidic bond.However, under appropriate conditions, they can be used to form thislinkage. Most glycosidases used for carbohydrate synthesis areexoglycosidases; the glycosyl transfer occurs at the non-reducingterminus of the substrate. The glycosidase binds a glycosyl donor in aglycosyl-enzyme intermediate that is either intercepted by water toyield the hydrolysis product, or by an acceptor, to generate a newglycoside or oligosaccharide. An exemplary pathway using anexoglycosidase is the synthesis of the core trisaccharide of allN-linked glycopeptides, including the β-mannoside linkage, which isformed by the action of β-mannosidase (Singh et al., Chem. Commun.993-994 (1996)).

[0019] In another exemplary application of the use of a glycosidase toform a glycosidic linkage, a mutant glycosidase has been prepared inwhich the normal nucleophilic amino acid within the active site ischanged to a non-nucleophilic amino acid. The mutant enzyme does nothydrolyze glycosidic linkages, but can still form them. Such a mutantglycosidase is used to prepare oligosaccharides using an α-glycosylfluoride donor and a glycoside acceptor molecule (Withers et al., U.S.Pat. No. 5,716,812).

[0020] Although their use is less common than that of theexoglycosidases, endoglycosidases are also utilized to preparecarbohydrates. Methods based on the use of endoglycosidases have theadvantage that an oligosaccharide, rather than a monosaccharide, istransferred. Oligosaccharide fragments have been added to substratesusing endo-β-N-acetylglucosamines such as endo-F, endo-M (Wang et al.,Tetrahedron Lett. 37: 1975-1978); and Haneda et al., Carbohydr. Res.292: 61-70 (1996)).

[0021] In addition to their use in preparing carbohydrates, the enzymesdiscussed above are applied to the synthesis of glycopeptides as well.The synthesis of a homogenous glycoform of ribonuclease B has beenpublished (Witte K. et al., J. Am. Chem. Soc. 119: 2114-2118 (1997)).The high mannose core of ribonuclease B was cleaved by treating theglycopeptide with endoglycosidase H. The cleavage occurred specificallybetween the two core GlcNAc residues. The tetrasaccharide sialyl Lewis Xwas then enzymatically rebuilt on the remaining GlcNAc anchor site onthe now homogenous protein by the sequential use ofβ-1,4-galactosyltransferase, α-2,3-sialyltransferase andα-1,3-fucosyltransferase V. However, while each enzymatically catalyzedstep proceeded in excellent yield, such procedures have not been adaptedfor the generation of glycopeptides on an industrial scale.

[0022] Methods combining both chemical and enzymatic synthetic elementsare also known in the art. For example, Yamamoto and coworkers(Carbohydr. Res. 305: 415-422 (1998)) reported the chemoenzymaticsynthesis of the glycopeptide, glycosylated Peptide T, using anendoglycosidase. The N-acetylglucosaminyl peptide was synthesized bypurely chemical means. The peptide was subsequently enzymaticallyelaborated with the oligosaccharide of human transferrin peptide. Thesaccharide portion was added to the peptide by treating it with anendo-β-N-acetylglucosaminidase. The resulting glycosylated peptide washighly stable and resistant to proteolysis when compared to the peptideT and N-acetylglucosaminyl peptide T.

[0023] The use of glycosyltransferases to modify peptide structure withreporter groups has been explored. For example, Brossmer et al. (U.S.Pat. No. 5,405,753) discloses the formation of a fluorescent-labeledcytidine monophosphate (“CMP”) derivative of sialic acid and the use ofthe fluorescent glycoside in an assay for sialyl transferase activityand for the fluorescent-labeling of cell surfaces, glycoproteins andpeptides. Gross et al. (Analyt. Biochem. 186: 127 (1990)) describe asimilar assay. Bean et al. (U.S. Pat. No. 5,432,059) discloses an assayfor glycosylation deficiency disorders utilizing reglycosylation of adeficiently glycosylated protein. The deficient protein isreglycosylated with a fluorescent-labeled CMP glycoside. Each of thefluorescent sialic acid derivatives is substituted with the fluorescentmoiety at either the 9-position or at the amine that is normallyacetylated in sialic acid. The methods using the fluorescent sialic acidderivatives are assays for the presence of glycosyltransferases or fornon-glycosylated or improperly glycosylated glycoproteins. The assaysare conducted on small amounts of enzyme or glycoprotein in a sample ofbiological origin. The enzymatic derivatization of a glycosylated ornon-glycosylated peptide on a preparative or industrial scale using amodified sialic acid has not been disclosed or suggested in the priorart.

[0024] Considerable effort has also been directed towards themodification of cell surfaces by altering glycosyl residues presented bythose surfaces. For example, Fukuda and coworkers have developed amethod for attaching glycosides of defined structure onto cell surfaces.The method exploits the relaxed substrate specificity of afucosyltransferase that can transfer fucose and fucose analogs bearingdiverse glycosyl substrates (Tsuboi et al., J. Biol. Chem. 271: 27213(1996)).

[0025] Enzymatic methods have also been used to activate glycosylresidues on a glycopeptide towards subsequent chemical elaboration. Theglycosyl residues are typically activated using galactose oxidase, whichconverts a terminal galactose residue to the corresponding aldehyde. Thealdehyde is subsequently coupled to an amine-containing modifying group.For example, Casares et al. (Nature Biotech. 19: 142 (2001)) haveattached doxorubicin to the oxidized galactose residues of a recombinantMHCII-peptide chimera.

[0026] Glycosyl residues have also been modified to contain ketonegroups. For example, Mahal and co-workers (Science 276: 1125 (1997))have prepared N-levulinoyl mannosamine (“ManLev”), which has a ketonefunctionality at the position normally occupied by the acetyl group inthe natural substrate. Cells were treated with the ManLev, therebyincorporating a ketone group onto the cell surface. See, also Saxon etal., Science 287: 2007 (2000); Hang et al., J. Am. Chem. Soc. 123: 1242(2001); Yarema et al., J. Biol. Chem. 273: 31168 (1998); and Charter etal., Glycobiology 10: 1049 (2000).

[0027] The methods of modifying cell surfaces have not been applied inthe absence of a cell to modify a glycosylated or non-glycosylatedpeptide. Further, the methods of cell surface modification are notutilized for the enzymatic incorporation preformed modified glycosyldonor moiety into a peptide. Moreover, none of the cell surfacemodification methods are practical for producing glycosyl-modifiedpeptides on an industrial scale.

[0028] Despite the efforts directed toward the enzymatic elaboration ofsaccharide structures, there remains still a need for an industriallypractical method for the modification of glycosylated andnon-glycosylated peptides with modifying groups such as water-solublepolymers, therapeutic moieties, biomolecules and the like. Of particularinterest are methods in which the modified peptide has improvedproperties, which enhance its use as a therapeutic or diagnostic agent.The present invention fulfills these and other needs.

SUMMARY OF THE INVENTION

[0029] The invention includes a multitude of methods of remodeling apeptide to have a specific glycan structure attached thereto. Althoughspecific glycan structures are described herein, the invention shouldnot be construed to be limited to any one particular structure. Inaddition, although specific peptides are described herein, the inventionshould not be limited by the nature of the peptide described, but rathershould encompass any and all suitable peptides and variations thereof.

[0030] The description which follows discloses the preferred embodimentsof the invention and provides a written description of the claimsappended hereto. The invention encompasses any and all variations ofthese embodiments that are or become apparent following a reading of thepresent specification.

[0031] The invention includes a cell-free, in vitro method of remodelinga granulocyte colony stimulating factor (G-CSF) peptide, the peptidehaving the formula:

[0032] wherein

[0033] AA is a terminal or internal amino acid residue of the peptide;

[0034] X¹—X² is a saccharide covalently linked to the AA, wherein

[0035] X¹ is a first glycosyl residue; and

[0036] X² is a second glycosyl residue covalently linked to X¹, whereinX¹ and X² are selected from monosaccharyl and oligosaccharyl residues;

[0037] the method comprising:

[0038] (a) removing X² or a saccharyl subunit thereof from the peptide,thereby forming a truncated glycan; and

[0039] (b) contacting the truncated glycan with at least oneglycosyltransferase and at least one glycosyl donor under conditionssuitable to transfer the at least one glycosyl donor to the truncatedglycan, thereby remodeling the G-CSF peptide.

[0040] In one embodiment, the method further comprises:

[0041] (c) removing X¹, thereby exposing the AA; and

[0042] (d) contacting the AA with at least one glycosyltransferase andat least one glycosyl donor under conditions suitable to transfer the atleast one glycosyl donor to the AA, thereby remodeling the G-CSFpeptide.

[0043] In another embodiment, the method comprises:

[0044] (e) prior to step (b), removing a group added to the saccharideduring post-translational modification.

[0045] In a preferred embodiment, the group is a member selected fromphosphate, sulfate, carboxylate and esters thereof.

[0046] In another embodiment of the invention, the peptide has theformula:

[0047] wherein

[0048] Z is a member selected from O, S, NH and a crosslinker.

[0049] In one embodiment, at least one of the glycosyl donors comprisesa modifying group. In another embodiment, the modifying group is amember selected from the group consisting of a polymer, a therapeuticmoiety, a detectable label, a reactive linker group, a targeting moiety,and a peptide. In a preferred embodiment, the modifying group is a watersoluble polymer. Preferably, the water soluble polymer comprisespoly(ethylene glycol). More preferably, the poly(ethylene glycol) has amolecular weight distribution that is essentially homodisperse.

[0050] Also included is a cell-free in vitro method of remodeling aG-CSF peptide, the peptide having the formula:

[0051] wherein

[0052] X³, X⁴, X⁵, X⁶, X⁷, and X¹⁷ are independently selectedmonosaccharyl or oligosaccharyl residues; and

[0053] a, b, c, d, e and x are independently selected from the integers0, 1 and 2, with the proviso that at least one member selected from a,b, c, d, and e and x are 1 or 2; the method comprising:

[0054] (a) removing at least one of X³, X⁴, X⁵, X⁶, X⁷, or X¹⁷, asaccharyl subunit thereof from the peptide, thereby forming a truncatedglycan; and

[0055] (b) contacting the truncated glycan with at least oneglycosyltransferase and at least one glycosyl donor under conditionssuitable to transfer the at least one glycosyl donor to the truncatedglycan, thereby remodeling the G-CSF peptide.

[0056] In one embodiment, the removing of step (a) produces a truncatedglycan in which a, b, c, e and x are each 0.

[0057] In another embodiment, X³, X⁵, and X⁷, are selected from thegroup consisting of (mannose)_(z) and (mannose)_(z)-(X⁸)_(y)

[0058] wherein

[0059] X⁸ is a glycosyl moiety selected from mono- andoligo-saccharides;

[0060] y is an integer selected from 0 and 1; and

[0061] z is an integer between 1 and 20, wherein

[0062] when z is 3 or greater, (mannose)_(z) is selected from linear andbranched structures.

[0063] In yet a further embodiment, X⁴ is selected from the groupconsisting of GlcNAc and xylose.

[0064] In an additional embodiment, wherein X³, X⁵, and X⁷ are(mannose)_(u), wherein

[0065] u is selected from the integers between 1 and 20, and when u is 3or greater, (mannose)_(u) is selected from linear and branchedstructures.

[0066] Preferably, at least one of the glycosyl donors comprises amodifying group. Also preferably, the modifying group is a memberselected from the group consisting of a polymer, a therapeutic moiety, adetectable label, a reactive linker group, a targeting moiety, and apeptide. Preferably, the modifying group is a water soluble polymer.Also preferably, the water soluble polymer comprises poly(ethyleneglycol). Preferably, the poly(ethylene glycol) has a molecular weightdistribution that is essentially homodisperse.

[0067] Further included in the invention is a cell-free in vitro methodof remodeling a G-CSF peptide comprising a glycan having the formula:

[0068] wherein

[0069] r, s, and t are integers independently selected from 0 and 1,

[0070] the method comprising:

[0071] (a) contacting the peptide with at least one glycosyltransferaseand at least one glycosyl donor under conditions suitable to transferthe at least one glycosyl donor to the glycan, thereby remodeling theG-CSF peptide.

[0072] In one embodiment, at least one of the glycosyl donors comprisesa modifying group. In another embodiment, the modifying group is amember selected from the group consisting of a polymer, a therapeuticmoiety, a detectable label, a reactive linker group, a targeting moiety,and a peptide. In an additional embodiment, the modifying group is awater soluble polymer. Preferably, the water soluble polymer comprisespoly(ethylene glycol). Also preferably, the poly(ethylene glycol) has amolecular weight distribution that is essentially homodisperse.

[0073] In one aspect of the invention, the peptide has the formula:

[0074] wherein

[0075] X⁹ and X¹⁰ are independently selected monosaccharyl oroligosaccharyl residues; and

[0076] m, n and f are integers selected from 0 and 1.

[0077] In another aspect, the peptide has the formula:

[0078] wherein

[0079] X¹¹ and X¹² are independently selected glycosyl moieties; and

[0080] r and x are integers independently selected from 0 and 1.

[0081] In one embodiment, X¹¹ and X¹² are (mannose)_(q), wherein

[0082] q is selected from the integers between 1 and 20, and when q isthree or greater, (mannose)_(q) is selected from linear and branchedstructures.

[0083] In a further aspect of the invention, the peptide has theformula:

[0084] wherein

[0085] X¹³, X¹⁴, and X¹⁵ are independently selected glycosyl residues;and

[0086] g, h, i, j, k, and p are independently selected from the integers0 and 1, with the proviso that at least one of g, h, i, j, k and p is 1.

[0087] In one embodiment,

[0088] X¹⁴ and X¹⁵ are members independently selected from GlcNAc andSia; and i and k are independently selected from the integers 0 and 1,with the proviso that at least one of i and k is 1 and if k is 1, g, hand j are 0.

[0089] In another aspect of the invention, the peptide has the formula:

[0090] wherein

[0091] X¹⁶ is a member selected from:

[0092] wherein

[0093] s and i are integers independently selected from 0 and 1.

[0094] Preferably, the removing utilizes a glycosidase.

[0095] There is also included a cell-free, in vitro method of remodelinga G-CSF peptide having the formula:

[0096] wherein

[0097] AA is a terminal or internal amino acid residue of the peptide;

[0098] X¹ is a glycosyl residue covalently linked to the AA, selectedfrom monosaccharyl and oligosaccharyl residues; and

[0099] u is an integer selected from 0 and 1,

[0100] the method comprising:

[0101] contacting the peptide with at least one glycosyltransferase andat least one glycosyl donor under conditions suitable to transfer the atleast one glycosyl donor to the truncated glycan, wherein the glycosyldonor comprises a modifying group, thereby remodeling the G-CSF peptide.

[0102] In one embodiment, the modifying group is a member selected fromthe group consisting of a polymer, a therapeutic moiety, a detectablelabel, a reactive linker group, a targeting moiety, and a peptide. Inanother embodiment, the modifying group is a water soluble polymer. In afurther embodiment, the water soluble polymer comprises poly(ethyleneglycol). Preferably, the poly(ethylene glycol) has a molecular weightdistribution that is essentially homodisperse.

[0103] There is also included a covalent conjugate between a G-CSFpeptide and a modifying group that alters a property of the peptide,wherein the modifying group is covalently attached to the peptide at apreselected glycosyl or amino acid residue of the peptide via an intactglycosyl linking group.

[0104] In one embodiment, the modifying group is a member selected fromthe group consisting of a polymer, a therapeutic moiety, a detectablelabel, a reactive linker group, a targeting moiety, and a peptide.

[0105] In another embodiment, the modifying group and an intact glycosyllinking group precursor are linked as a covalently attached unit to thepeptide via the action of an enzyme, the enzyme converting the precursorto the intact glycosyl linking group, thereby forming the conjugate.

[0106] In a further embodiment, the covalent conjugate comprises a firstmodifying group covalently linked to a first residue of the peptide viaa first intact glycosyl linking group, and a second glycosyl linkinggroup linked to a second residue of the peptide via a second intactglycosyl linking group. Preferably, the first residue and the secondresidue are structurally identical. Also preferably, the first residueand the second residue have different structures. Additionallypreferably, the first residue and the second residue are glycosylresidues. Also preferably, the first residue and the second residue areamino acid residues. Further preferably, the peptide is remodeled priorto forming the conjugate. In addition preferably, the remodeled peptideis remodeled to introduce an acceptor moiety for the intact glycosyllinking group. Also preferably, the modifying group is a water-solublepolymer. Further preferably, the water-soluble polymer comprisespoly(ethylene glycol). Also preferably, the intact glycosyl linking unitis a member selected from the group consisting of a sialic acid residue,a Gal residue, a GlcNAc residue and a GalNAc residue. Preferably, thepoly(ethylene glycol) has a molecular weight distribution that isessentially homodisperse.

[0107] There is further included a method of forming a covalentconjugate between a polymer and a glycosylated or non-glycosylatedpeptide, wherein the polymer is conjugated to the peptide via an intactglycosyl linking group interposed between and covalently linked to boththe peptide and the polymer, the method comprising:

[0108] contacting the peptide with a mixture comprising a nucleotidesugar covalently linked to the polymer and a glycosyltransferase forwhich the nucleotide sugar is a substrate under conditions sufficient toform the conjugate, wherein the peptide is G-CSF.

[0109] In one embodiment, the polymer is a water-soluble polymer. Inanother embodiment, the glycosyl linking group is covalently attached toa glycosyl residue covalently attached to the peptide. In a furtherembodiment, the glycosyl linking group is covalently attached to anamino acid residue of the peptide. Further, the polymer comprises amember selected from the group consisting of a polyalkylene oxide and apolypeptide. In addition, the polyalkylene oxide is poly(ethyleneglycol). Preferably, the poly(ethylene glycol) has a degree ofpolymerization of from about 1 to about 20,000. Further preferably, thepolyethylene glycol has a degree of polymerization of from about 1 toabout 5,000. In addition preferably, the polyethylene glycol has adegree of polymerization of from about 1 to about 1,000.

[0110] The method also includes wherein the glycosyltransferase isselected from the group consisting of sialyltransferase,galactosyltransferase, glucosyltransferase, GalNAc transferase, GlcNActransferase, fucosyltransferase, and mannosyltransferase. Preferably,the glycosyltransferase is recombinantly produced. Also preferably, theglycosyltransferase is a recombinant prokaryotic enzyme or a recombinanteukaryotic enzyme. Further preferably, the nucleotide sugar is selectedfrom the group consisting of UDP-glycoside, CMP-glycoside, andGDP-glycoside. In addition preferably, the nucleotide sugar is selectedfrom the group consisting of UDP-galactose, UDP-galactosamine,UDP-glucose, UDP-glucosamine, UDP-N-acetylgalactosamine,UDP-N-acetylglucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid,CMP-NeuAc. Also preferably, the glycosylated peptide is partiallydeglycosylated prior to the contacting. Further, the intact glycosyllinking group is a sialic acid residue, and even further, the method isperformed in a cell-free environment.

[0111] In one aspect, the covalent conjugate is isolated. In anotheraspect, the covalent conjugate is isolated by membrane filtration.

[0112] There is further provided a composition for forming a conjugatebetween a peptide and a modified sugar, the composition comprising: anadmixture of a modified sugar, a glycosyltransferase, and a peptideacceptor substrate, wherein the modified sugar has covalently attachedthereto a member selected from a polymer, a therapeutic moiety and abiomolecule, wherein the peptide is G-CSF.

[0113] In addition, there is provided a G-CSF peptide remodeled by themethods of the invention and pharmaceutical compositions comprising theG-CSF peptides.

[0114] The invention also includes a cell-free, in vitro method ofremodeling a peptide having the formula:

[0115] wherein

[0116] AA is a terminal or internal amino acid residue of the peptide,

[0117] the method comprising:

[0118] contacting the peptide with at least one glycosyltransferase andat least one glycosyl donor under conditions suitable to transfer the atleast one glycosyl donor to the amino acid residue, wherein the glycosyldonor comprises a modifying group, thereby remodeling the peptide,wherein the peptide is G-CSF.

[0119] In addition, the invention includes a method of forming aconjugate between a G-CSF peptide and a modifying group, wherein themodifying group is covalently attached to the G-CSF peptide through anintact glycosyl linking group, the G-CSF peptide comprising a glycosylresidue having the formula:

[0120] wherein

[0121] a, b, c, and e are members independently selected from 0 and 1;

[0122] d is 0; and

[0123] R is a modifying group, a sialic acid or an oligosialic acid,

[0124] the method comprising:

[0125] (a) contacting the G-CSF peptide with a glycosyltransferase and amodified glycosyl donor, comprising a glycosyl moiety which is asubstrate for the glycosyltransferase covalently linked to the modifyinggroup, under conditions appropriate for the formation of the intactglycosyl linking group.

[0126] In one embodiment, the method further comprises:

[0127] (b) prior to step (a), contacting the G-CSF peptide with asialidase under conditions appropriate to remove sialic acid from theG-CSF peptide.

[0128] In another embodiment, the method further comprises:

[0129] (c) prior to step (a), contacting the G-CSF peptide with agalactosyl transferase and a galactose donor under conditionsappropriate to transfer the galactose to the G-CSF peptide.

[0130] In yet another embodiment, the method further comprises:

[0131] (d) contacting the product from step (a) with a moiety thatreacts with the modifying group, thereby forming a conjugate between theintact glycosyl linking group and the moiety.

[0132] Additionally, the method also comprises:

[0133] (e) prior to step (a), contacting the G-CSF peptide withN-acetylgalactosamine transferase and a GalNAc donor under conditionsappropriate to transfer GalNAc to the G-CSF peptide.

[0134] Further, the method comprises:

[0135] (f) prior to step (a), contacting the G-CSF peptide withendo-N-acetylgalactosaminidase operating synthetically and a GalNAcdonor under conditions appropriate to transfer GalNAc to the G-CSFpeptide.

[0136] In one aspect, wherein the modifying group is a member selectedfrom a polymer, a toxin, a radioisotope, a therapeutic moiety and aglycoconjugate.

[0137] The method further provides that

[0138] a, b, c, and e are 0.

[0139] The method also provides that

[0140] a and e are members independently selected from 0 and 1; and

[0141] b, c, and d are 0.

[0142] The method additionally provides that

[0143] a, b, c, d, and e are members independently selected from 0 and1.

[0144] Also included is a G-CSF peptide conjugate formed by the methodsof the invention.

[0145] there is also included a G-CSF peptide comprising one or moreglycans, having a glycoconjugate molecule covalently attached to thepeptide. In one aspect, the one or more glycans is a monoantennaryglycan. In another aspect, the one or more glycans is a biantennaryglycan. In a further aspect, the one or more glycans is a triantennaryglycan. In yet a further aspect, the one or more glycans is at least atriantennary glycan. Additionally, the one or more glycans comprises atleast two glycans comprising a mixture of mono or multiantennaryglycans. Further, the one or more glycans is selected from an N-linkedglycan and an O-linked glycan. Also, the one or more glycans is at leasttwo glycans selected from an N-linked and an O-linked glycan. Inaddition, peptide is expressed in a cell selected from the groupconsisting of a prokaryotic cell and a eukaryotic cell, wherein theeukaryotic cell is selected from the group consisting of a mammaliancell, an insect cell and a yeast cell.

[0146] There is also included a method of treating a mammal having adisease selected from the group consisting of an infectious disease,acute myeloid leukemia, non-myeloid cancer, chronic or persistentneutropenia, the method comprising administering to the mammal a G-CSFpeptide having one or more glycans having a glycoconjugate moleculeattached to the peptide. Preferably, the infectious disease is selectedfrom the group consisting of a bacterial and a viral disease. Alsopreferably, wherein the glycoconjugate molecule is poly(ethyleneglycol), and the mammal is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

[0147] For the purpose of illustrating the invention, there are depictedin the drawings certain embodiments of the invention. However, theinvention is not limited to the precise arrangements andinstrumentalities of the embodiments depicted in the drawings.

[0148]FIG. 1 is a scheme depicting a trimannosyl core glycan (left side)and the enzymatic process for the generation of a glycan having abisecting GlcNAc (right side).

[0149]FIG. 2 is a scheme depicting an elemental trimannosyl corestructure and complex chains in various degrees of completion. The invitro enzymatic generation of an elemental trimannosyl core structurefrom a complex carbohydrate glycan structure which does not contain abisecting GlcNAc residue is shown, as is the generation of a glycanstructure therefrom which contains a bisecting GlcNAc. Symbols: squares:GlcNAc; light circles: Man; dark circles: Gal; triangles: NeuAc.

[0150]FIG. 3 is a scheme for the enzymatic generation of a sialylatedglycan structure (right side) beginning with a glycan having atrimannosyl core and a bisecting GlcNAc (left side).

[0151]FIG. 4 is a scheme of a typical high mannose containing glycanstructure (left side) and the enzymatic process for reduction of thisstructure to an elemental trimannosyl core structure. In this scheme, Xis mannose as a monosaccharide, an oligosaccharide or a polysaccharide.

[0152]FIG. 5 is a diagram of a fucose and xylose containing N-linkedglycan structure typically produced in plant cells.

[0153]FIG. 6 is a diagram of a fucose containing N-linked glycanstructure typically produced in insect cells. Note that the glycan mayhave no core fucose, it amy have a single core fucose with eitherlinkage, or it may have a single core fucose having a preponderance ofone linkage.

[0154]FIG. 7 is a scheme depicting a variety of pathways for thetrimming of a high mannose structure and the synthesis of complex sugarchains therefrom. Symbols: squares: GlcNAc; circles: Man; diamonds:fucose; pentagon: xylose.

[0155]FIG. 8 is a scheme depicting in vitro strategies for the synthesisof complex structures from an elemental trimannosyl core structure.Symbols: Squares: GlcNAc; light circles: Man; dark circles: Gal; darktriangles: NeuAc; GnT: N-acetyl glucosaminyltransferase; GalT:galactosyltransferase; ST: sialyltransferase.

[0156]FIG. 9 is a scheme depicting two in vitro strategies for thesynthesis of monoantennary glycans, and the optional glycoPEGylation ofthe same. Dark squares: GlcNAc; dark circles: Man; light circles: Gal;dark triangles: sialic acid.

[0157]FIG. 10 is a scheme depicting two in vitro strategies for thesynthesis of monoantennary glycans, and the optional glycoPEGylation ofthe same. Dark squares: GlcNAc; dark circles: Man; light circles: Gal;dark triangles: sialic acid.

[0158]FIG. 11 is a scheme depicting various complex structures, whichmay be synthesized from an elemental trimannosyl core structure.Symbols: Squares: GlcNAc; light circles: Man; dark circles: Gal;triangles: NeuAc; diamonds: fucose; FT and FucT: fucosyltransferase;GalT: galactosyltransferase; ST: sialyltransferase; Le: Lewis antigen;SLe: sialylated Lewis antigen.

[0159]FIG. 12 is an exemplary scheme for preparing O-linkedglycopeptides originating with serine or threonine. Optionally, a watersoluble polymer (WSP) such as poly(ethylene glycol) is added to thefinal glycan structure.

[0160]FIG. 13 is a series of diagrams depicting the four types ofO-glycan structures, termed cores 1 through 4. The core structure isoutlined in dotted lines.

[0161]FIG. 14, comprising FIG. 14A and FIG. 14B, is a series of schemesshowing an exemplary embodiment of the invention in which carbohydrateresidues comprising complex carbohydrate structures and/or high mannosehigh mannose structures are trimmed back to the first generationbiantennary structure. Optionally, fucose is added only after reactionwith GnT I. A modified sugar bearing a water-soluble polymer (WSP) isthen conjugated to one or more of the sugar residues exposed by thetrimming back process.

[0162]FIG. 15 is a scheme similar to that shown in FIG. 4, in which ahigh mannose or complex structure is “trimmed back” to the mannosebeta-linked core and a modified sugar bearing a water soluble polymer isthen conjugated to one or more of the sugar residues exposed by thetrimming back process. Sugars are added sequentially usingglycosyltransferases.

[0163]FIG. 16 is a scheme similar to that shown in FIG. 4, in which ahigh mannose or complex structure is trimmed back to the GlcNAc to whichthe first mannose is attached, and a modified sugar bearing a watersoluble polymer is then conjugated to one or more of the sugar residuesexposed by the trimming back process. Sugars are added sequentiallyusing glycosyltransferases.

[0164]FIG. 17 is a scheme similar to that shown in FIG. 4, in which ahigh mannose or cpomplex structure is trimmed back to the first GlcNAcattached to the Asn of the peptide, following which a water solublepolymer is conjugated to one or more sugar residues which havesubsequently been added on. Sugars are added sequentially usingglycosyltransferases.

[0165]FIG. 18, comprising FIGS. 18A and 18B, is a scheme in which anN-linked carbohydrate is optionally trimmed back from a high mannose orcpmplex structure, and subsequently derivatized with a modified sugarmoiety (Gal or GlcNAc) bearing a water-soluble polymer.

[0166]FIG. 19, comprising FIGS. 19A and 19B, is a scheme in which anN-linked carbohydrate is trimmed back from a high mannose or complexstructure and subsequently derivatized with a sialic acid moiety bearinga water-soluble polymer. Sugars are added sequentially usingglycosyltransferases.

[0167]FIG. 20 is a scheme in which an N-linked carbohydrate isoptionally trimmed back from a high mannose oor complex structure andsubsequently derivatized with one or more sialic acid moieties, andterminated with a sialic acid derivatized with a water-soluble polymer.Sugars are added sequentially using glycosyltransferases.

[0168]FIG. 21 is a scheme in which an O-linked saccharide is “trimmedback” and subsequently conjugated to a modified sugar bearing awater-soluble polymer. In the exemplary scheme, the carbohydrate moietyis “trimmed back” to the first generation of the biantennary structure.

[0169]FIG. 22 is an exemplary scheme for trimming back the carbohydratemoiety of an O-linked glycopeptide to produce a mannose available forconjugation with a modified sugar having a water-soluble polymerattached thereto.

[0170]FIG. 23, comprising FIG. 23A to FIG. 23C, is a series of exemplaryschemes. FIG. 23A is a scheme that illustrates addition of a PEGylatedsugar, followed by the addition of a non-modified sugar. FIG. 23B is ascheme that illustrates the addition of more that one kind of modifiedsugar onto one glycan. FIG. 23C is a scheme that illustrates theaddition of different modified sugars onto O-linked glycans and N-linkedglycans.

[0171]FIG. 24 is a diagram of various methods of improving thetherapeutic function of a peptide by glycan remodeling, includingconjugation.

[0172]FIG. 25 is a set of schemes for glycan remodeling of a therapeuticpeptide to treat Gaucher Disease.

[0173]FIG. 26 is a scheme for glycan remodeling to generate glycanshaving a terminal mannose-6-phosphate moiety.

[0174]FIG. 27 is a diagram illustrating the array of glycan structuresfound on CHO-produced glucocerebrosidase (Cerezyme™) after sialylation.

[0175]FIG. 28, comprising FIG. 28A to FIG. 28Z and FIG. 28AA to FIG.28CC, is a list of peptides useful in the methods of the invention.

[0176]FIG. 29, comprising FIGS. 29A to 29G, provides exemplary schemesfor remodeling glycan structures on granulocyte colony stimulatingfactor (G-CSF). FIG. 29A is a diagram depicting the G-CSF peptideindicating the amino acid residue to which a glycan is bonded, and anexemplary glycan formula linked thereto. FIGS. 29B to 29G are diagramsof contemplated remodeling steps of the glycan of the peptide in FIG.29A based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure.

[0177]FIG. 30, comprising FIGS. 30A to 30EE sets forth exemplary schemesfor remodeling glycan structures on interferon-alpha. FIG. 30A is adiagram depicting the interferon-alpha isoform ¹⁴C peptide indicatingthe amino acid residue to which a glycan is bonded, and an exemplaryglycan formula linked thereto. FIGS. 30B to 30D are diagrams ofcontemplated remodeling steps of the glycan of the peptide in FIG. 30Abased on the type of cell the peptide is expressed in and the desiredremodeled glycan structure. FIG. 30E is a diagram depicting theinterferon-alpha isoform ¹⁴C peptide indicating the amino acid residueto which a glycan is linked, and an exemplary glycan formula linkedthereto. FIGS. 30F to 30N are diagrams of contemplated remodeling stepsof the glycan of the peptide in FIG. 30E based on the type of cell thepeptide is expressed in and the desired remodeled glycan structure. FIG.30O is a diagram depicting the interferon-alpha isoform 2a or 2bpeptides indicating the amino acid residue to which a glycan is linked,and an exemplary glycan formula linked thereto. FIGS. 30P to 30W arediagrams of contemplated remodeling steps of the glycan of the peptidein FIG. 30O based on the type of cell the peptide is expressed in andthe desired remodeled glycan structure. FIG. 30X is a diagram depictingthe interferon-alpha-mucin fusion peptides indicating the residue(s)which is linked to glycans contemplated for remodeling, and exemplaryglycan formulas linked thereto. FIGS. 30Y to 30AA are diagrams ofcontemplated remodeling steps of the glycan of the peptides in FIG. 30Xbased on the type of cell the peptide is expressed in and the desiredremodeled glycan structure. FIG. 30BB is a diagram depicting theinterferon-alpha-mucin fusion peptides and interferon-alpha peptidesindicating the residue(s) which bind to glycans contemplated forremodeling, and formulas for the glycans. FIGS. 30CC to 30EE arediagrams of contemplated remodeling steps of the glycan of the peptidesin FIG. 30BB based on the type of cell the peptide is expressed in andthe desired remodeled glycan structure.

[0178]FIG. 31, comprising FIGS. 31A to 31S, sets forth exemplary schemesfor remodeling glycan structures on interferon-beta. FIG. 31A is adiagram depicting the interferon-beta peptide indicating the amino acidresidue to which a glycan is linked, and an exemplary glycan formulalinked thereto. FIGS. 31B to 31O are diagrams of contemplated remodelingsteps of the glycan of the peptide in FIG. 31A based on the type of cellthe peptide is expressed in and the desired remodeled glycan structure.FIG. 31P is a diagram depicting the interferon-beta peptide indicatingthe amino acid residue to which a glycan is linked, and an exemplaryglycan formula linked thereto. FIGS. 31Q to 31S are diagrams ofcontemplated remodeling steps of the glycan of the peptide in FIG. 31Pbased on the type of cell the peptide is expressed in and the desiredremodeled glycan structure.

[0179]FIG. 32, comprising FIGS. 32A to 32D, sets forth exemplary schemesfor remodeling glycan structures on Factor VII and Factor VIIa. FIG. 32Ais a diagram depicting the Factor-VII and Factor-VIIa peptides A (solidline) and B (dotted line) indicating the residues which bind to glycanscontemplated for remodeling, and the formulas for the glycans. FIGS. 32Bto 32D are diagrams of contemplated remodeling steps of the glycan ofthe peptide in FIG. 32A based on the type of cell the peptide isexpressed in and the desired remodeled glycan structure.

[0180]FIG. 33, comprising FIGS. 33A to 33G, sets forth exemplary schemesfor remodeling glycan structures on Factor IX. FIG. 33A is a diagramdepicting the Factor-IX peptide indicating residues which bind toglycans contemplated for remodeling, and formulas of the glycans. FIGS.33B to 33G are diagrams of contemplated remodeling steps of the glycanof the peptide in FIG. 33A based on the type of cell the peptide isexpressed in and the desired remodeled glycan structure.

[0181]FIG. 34, comprising FIGS. 34A to 34J, sets forth exemplary schemesfor remodeling glycan structures on follicle stimulating hormone (FSH),comprising α and β subunits. FIG. 34A is a diagram depicting theFollicle Stimulating Hormone peptides FSHα and FSHβ indicating theresidues which bind to glycans contemplated for remodeling, andexemplary glycan formulas linked thereto. FIGS. 34B to 34J are diagramsof contemplated remodeling steps of the glycan of the peptides in FIG.34A based on the type of cell the peptides are expressed in and thedesired remodeled glycan structures.

[0182]FIG. 35, comprising FIGS. 35A to 35AA, sets forth exemplaryschemes for remodeling glycan structures on Erythropoietin (EPO). FIG.35A is a diagram depicting the EPO peptide indicating the residues whichbind to glycans contemplated for remodeling, and formulas for theglycans. FIGS. 35B to 35S are diagrams of contemplated remodeling stepsof the glycan of the peptide in FIG. 35A based on the type of cell thepeptide is expressed in and the desired remodeled glycan structure. FIG.35T is a diagram depicting the EPO peptide indicating the residues whichbind to glycans contemplated for remodeling, and formulas for theglycans. FIGS. 35U to 35W are diagrams of contemplated remodeling stepsof the glycan of the peptide in FIG. 35T based on the type of cell thepeptide is expressed in and the desired remodeled glycan structure. FIG.35X is a diagram depicting the EPO peptide indicating the residues whichbind to glycans contemplated for remodeling, and formulas for theglycans. FIGS. 35Y to 35AA are diagrams of contemplated remodeling stepsof the glycan of the peptide in FIG. 35X based on the type of cell thepeptide is expressed in and the desired remodeled glycan structure.

[0183]FIG. 36, comprising FIGS. 36A to 36K sets forth exemplary schemesfor remodeling glycan structures on Granulocyte-Macrophage ColonyStimulating Factor (GM-CSF). FIG. 36A is a diagram depicting the GM-CSFpeptide indicating the residues which bind to glycans contemplated forremodeling, and formulas for the glycans. FIGS. 36B to 36G are diagramsof contemplated remodeling steps of the glycan of the peptide in FIG.36A based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure. FIG. 36H is a diagram depicting theGM-CSF peptide indicating the residues which bind to glycanscontemplated for remodeling, and formulas for the glycans. FIGS. 36I to36K are diagrams of contemplated remodeling steps of the glycan of thepeptide in FIG. 36H based on the type of cell the peptide is expressedin and the desired remodeled glycan structure.

[0184]FIG. 37, comprising FIGS. 37A to 37N, sets forth exemplary schemesfor remodeling glycan structures on interferon-gamma. FIG. 37A is adiagram depicting an interferon-gamma peptide indicating the residueswhich bind to glycans contemplated for remodeling, and exemplary glycanformulas linked thereto. FIGS. 37B to 37G are diagrams of contemplatedremodeling steps of the peptide in FIG. 37A based on the type of cellthe peptide is expressed in and the desired remodeled glycan structure.FIG. 37H is a diagram depicting an interferon-gamma peptide indicatingthe residues which bind to glycans contemplated for remodeling, andexemplary glycan formulas linked thereto. FIGS. 37I to 37N are diagramsof contemplated remodeling steps of the peptide in FIG. 37H based on thetype of cell the peptide is expressed in and the desired remodeledglycan structure.

[0185]FIG. 38, comprising FIGS. 38A to 38N, sets forth exemplary schemesfor remodeling glycan structures on α₁-antitrypsin (ATT, or α-1 proteaseinhibitor). FIG. 38A is a diagram depicting an AAT peptide indicatingthe residues which bind to glycans contemplated for remodeling, andexemplary glycan formulas linked thereto. FIGS. 38B to 38F are diagramsof contemplated remodeling steps of the glycan of the peptide in FIG.38A based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure. FIG. 38G is a diagram depicting anAAT peptide indicating the residues which bind to glycans contemplatedfor remodeling, and exemplary glycan formulas linked thereto. FIGS. 38Hto 38J are diagrams of contemplated remodeling steps of the peptide inFIG. 38G based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure. FIG. 38K is a diagram depicting anAAT peptide indicating the residues which bind to glycans contemplatedfor remodeling, and exemplary glycan formulas linked thereto. FIGS. 38Lto 38N are diagrams of contemplated remodeling steps of the peptide inFIG. 38K based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure.

[0186]FIG. 39, comprising FIGS. 39A to 39J sets forth exemplary schemesfor remodeling glycan structures on glucocerebrosidase. FIG. 39A is adiagram depicting the glucocerebrosidase peptide indicating the residueswhich bind to glycans contemplated for remodeling, and exemplary glycanformulas linked thereto. FIGS. 39B to 39F are diagrams of contemplatedremodeling steps of the glycan of the peptide in FIG. 39A based on thetype of cell the peptide is expressed in and the desired remodeledglycan structure. FIG. 39G is a diagram depicting the glucocerebrosidasepeptide indicating the residues which bind to glycans contemplated forremodeling, and exemplary glycan formulas linked thereto. FIGS. 39H to39K are diagrams of contemplated remodeling steps of the glycan of thepeptide in FIG. 39G based on the type of cell the peptide is expressedin and the desired remodeled glycan structure.

[0187]FIG. 40, comprising FIGS. 40A to 40W, sets forth exemplary schemesfor remodeling glycan structures on Tissue-Type Plasminogen Activator(TPA). FIG. 40A is a diagram depicting the TPA peptide indicating theresidues which bind to glycans contemplated for remodeling, and formulasfor the glycans. FIGS. 40B to 40G are diagrams of contemplatedremodeling steps of the peptide in FIG. 40A based on the type of cellthe peptide is expressed in and the desired remodeled glycan structure.FIG. 40H is a diagram depicting the TPA peptide indicating the residueswhich bind to glycans contemplated for remodeling, and formulas for theglycans. FIGS. 40I to 40K are diagrams of contemplated remodeling stepsof the peptide in FIG. 40H based on the type of cell the peptide isexpressed in and the desired remodeled glycan structure. FIG. 40L is adiagram depicting a mutant TPA peptide indicating the residues whichbind to glycans contemplated for remodeling, and the formula for theglycans. FIGS. 40M to 40O are diagrams of contemplated remodeling stepsof the peptide in FIG. 40L based on the type of cell the peptide isexpressed in and the desired remodeled glycan structure. FIG. 40P is adiagram depicting a mutant TPA peptide indicating the residues whichbind to glycans contemplated for remodeling, and formulas for theglycans. FIGS. 40Q to 40S are diagrams of contemplated remodeling stepsof the peptide in FIG. 40P based on the type of cell the peptide isexpressed in and the desired remodeled glycan structure. FIG. 40T is adiagram depicting a mutant TPA peptide indicating the residues whichlinks to glycans contemplated for remodeling, and formulas for theglycans. FIGS. 40U to 40W are diagrams of contemplated remodeling stepsof the peptide in FIG. 40T based on the type of cell the peptide isexpressed in and the desired remodeled glycan structure.

[0188]FIG. 41, comprising FIGS. 41A to 41G, sets forth exemplary schemesfor remodeling glycan structures on Interleukin-2 (IL-2). FIG. 41A is adiagram depicting the Interleukin-2 peptide indicating the amino acidresidue to which a glycan is linked, and an exemplary glycan formulalinked thereto. FIGS. 41B to 41G are diagrams of contemplated remodelingsteps of the glycan of the peptide in FIG. 41A based on the type of cellthe peptide is expressed in and the desired remodeled glycan structure.

[0189]FIG. 42, comprising FIGS. 42A to 42M, sets forth exemplary schemesfor remodeling glycan structures on Factor VIII. FIG. 42A are theformulas for the glycans that bind to the N-linked glycosylation sites(A and A′) and to the O-linked sites (B) of the Factor VIII peptides.FIGS. 42B to 42F are diagrams of contemplated remodeling steps of thepeptides in FIG. 42A based on the type of cell the peptide is expressedin and the desired remodeled glycan structure. FIG. 42G are the formulasfor the glycans that bind to the N-linked glycosylation sites (A and A′)and to the O-linked sites (B) of the Factor VIII peptides. FIGS. 42H to42M are diagrams of contemplated remodeling steps of the peptides inFIG. 42G based on the type of cell the peptide is expressed in and thedesired remodeled glycan structures.

[0190]FIG. 43, comprising FIGS. 43A to 43M, sets forth exemplary schemesfor remodeling glycan structures on urokinase. FIG. 43A is a diagramdepicting the urokinase peptide indicating a residue which is linked toa glycan contemplated for remodeling, and an exemplary glycan formulalinked thereto. FIGS. 43B to 43F are diagrams of contemplated remodelingsteps of the peptide in FIG. 43A based on the type of cell the peptideis expressed in and the desired remodeled glycan structure. FIG. 43G isa diagram depicting the urokinase peptide indicating a residue which islinked to a glycan contemplated for remodeling, and an exemplary glycanformula linked thereto. FIGS. 43H to 43L are diagrams of contemplatedremodeling steps of the peptide in FIG. 43G based on the type of cellthe peptide is expressed in and the desired remodeled glycan structure.

[0191]FIG. 44, comprising FIGS. 44A to 44J, sets forth exemplary schemesfor remodeling glycan structures on human DNase (hDNase). FIG. 44A is adiagram depicting the human DNase peptide indicating the residues whichbind to glycans contemplated for remodeling, and exemplary glycanformulas linked thereto. FIGS. 44B to 44F are diagrams of contemplatedremodeling steps of the peptide in FIG. 44A based on the type of cellthe peptide is expressed in and the desired remodeled glycan structure.FIG. 44G is a diagram depicting the human DNase peptide indicatingresidues which bind to glycans contemplated for remodeling, andexemplary glycan formulas linked thereto. FIGS. 44H to 44J are diagramsof contemplated remodeling steps of the peptide in FIG. 44F based on thetype of cell the peptide is expressed in and the desired remodeledglycan structure.

[0192]FIG. 45, comprising FIGS. 45A to 45L, sets forth exemplary schemesfor remodeling glycan structures on insulin. FIG. 45A is a diagramdepicting the insulin peptide mutated to contain an N glycosylation siteand an exemplary glycan formula linked thereto. FIGS. 45B to 45D arediagrams of contemplated remodeling steps of the peptide in FIG. 45Abased on the type of cell the peptide is expressed in and the desiredremodeled glycan structure. FIG. 45E is a diagram depictinginsulin-mucin fusion peptides indicating a residue(s) which is linked toa glycan contemplated for remodeling, and an exemplary glycan formulalinked thereto. FIGS. 45F to 45H are diagrams of contemplated remodelingsteps of the peptide in FIG. 45E based on the type of cell the peptideis expressed in and the desired remodeled glycan structure. FIG. 45I isa diagram depicting the insulin-mucin fusion peptides and insulinpeptides indicating a residue(s) which is linked to a glycancontemplated for remodeling, and formulas for the glycan. FIGS. 45J to45L are diagrams of contemplated remodeling steps of the peptide in FIG.45I based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure.

[0193]FIG. 46, comprising FIGS. 46A to 46K, sets forth exemplary schemesfor remodeling glycan structures on the M-antigen (preS and S) of theHepatitis B surface protein (HbsAg). FIG. 46A is a diagram depicting theM-antigen peptide indicating the residues which bind to glycanscontemplated for remodeling, and formulas for the glycans. FIGS. 46B to46G are diagrams of contemplated remodeling steps of the peptide in FIG.46A based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure. FIG. 46H is a diagram depicting theM-antigen peptide indicating the residues which bind to glycanscontemplated for remodeling, and formulas for the glycans. FIGS. 46I to46K are diagrams of contemplated remodeling steps of the peptide in FIG.46H based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure.

[0194]FIG. 47, comprising FIGS. 47A to 47K, sets forth exemplary schemesfor remodeling glycan structures on human growth hormone, including N, Vand variants thereof. FIG. 47A is a diagram depicting the human growthhormone peptide indicating a residue which is linked to a glycancontemplated for remodeling, and an exemplary glycan formula linkedthereto. FIGS. 47B to 47D are diagrams of contemplated remodeling stepsof the glycan of the peptide in FIG. 47A based on the type of cell thepeptide is expressed in and the desired remodeled glycan structure. FIG.47E is a diagram depicting the three fusion peptides comprising thehuman growth hormone peptide and part or all of a mucin peptide, andindicating a residue(s) which is linked to a glycan contemplated forremodeling, and exemplary glycan formula(s) linked thereto. FIGS. 47F to47K are diagrams of contemplated remodeling steps of the glycan of thepeptides in FIG. 47E based on the type of cell the peptide is expressedin and the desired remodeled glycan structure.

[0195]FIG. 48, comprising FIGS. 48A to 48G, sets forth exemplary schemesfor remodeling glycan structures on a TNF Receptor-IgG Fc region fusionprotein (Enbrel™). FIG. 48A is a diagram depicting a TNF Receptor—IgG Fcregion fusion peptide which may be mutated to contain additionalN-glycosylation sites indicating the residues which bind to glycanscontemplated for remodeling, and formulas for the glycans. The TNFreceptor peptide is depicted in bold line, and the IgG Fc regions isdepicted in regular line. FIGS. 48B to 48G are diagrams of contemplatedremodeling steps of the peptide in FIG. 48A based on the type of cellthe peptide is expressed in and the desired remodeled glycan structure.

[0196]FIG. 49, comprising FIGS. 49A to 49D, sets forth exemplary schemesfor remodeling glycan structures on an anti-HER2 monoclonal antibody(Herceptin™). FIG. 49A is a diagram depicting an anti-HER2 monoclonalantibody which has been mutated to contain an N-glycosylation site(s)indicating a residue(s) on the antibody heavy chain which is linked to aglycan contemplated for remodeling, and an exemplary glycan formulalinked thereto. FIGS. 49B to 49D are diagrams of contemplated remodelingsteps of the glycan of the peptides in FIG. 49A based on the type ofcell the peptide is expressed in and the desired remodeled glycanstructure.

[0197]FIG. 50, comprising FIGS. 50A to 50D, sets forth exemplary schemesfor remodeling glycan structures on a monoclonal antibody to Protein Fof Respiratory Syncytial Virus (Synagis™). FIG. 50A is a diagramdepicting a monoclonal antibody to Protein F peptide which is mutated tocontain an N-glycosylation site(s) indicating a residue(s) which islinked to a glycan contemplated for remodeling, and an exemplary glycanformula linked thereto. FIGS. 50B to 50D are diagrams of contemplatedremodeling steps of the peptide in FIG. 50A based on the type of cellthe peptide is expressed in and the desired remodeled glycan structure.

[0198]FIG. 51, comprising FIGS. 51A to 51D, sets forth exemplary schemesfor remodeling glycan structures on a monoclonal antibody to TNF-α(Remicade™). FIG. 51A is a diagram depicting a monoclonal antibody toTNF-α which has an N-glycosylation site(s) indicating a residue which islinked to a glycan contemplated for remodeling, and an exemplary glycanformula linked thereto. FIGS. 51B to 51D are diagrams of contemplatedremodeling steps of the peptide in FIG. 51A based on the type of cellthe peptide is expressed in and the desired remodeled glycan structure.

[0199]FIG. 52, comprising FIGS. 52A to 52L, sets forth exemplary schemesfor remodeling glycan structures on a monoclonal antibody toglycoprotein IIb/IIIa (Reopro™). FIG. 52A is a diagram depicting amutant monoclonal antibody to glycoprotein IIb/IIIa peptides which havebeen mutated to contain an N-glycosylation site(s) indicating theresidue(s) which bind to glycans contemplated for remodeling, andexemplary glycan formulas linked thereto. FIGS. 52B to 52D are diagramsof contemplated remodeling steps based on the type of cell the peptideis expressed in and the desired remodeled glycan structure. FIG. 52E isa diagram depicting monoclonal antibody to glycoprotein IIb/IIIa-mucinfusion peptides indicating the residues which bind to glycanscontemplated for remodeling, and exemplary glycan formulas linkedthereto. FIGS. 52F to 52H are diagrams of contemplated remodeling stepsbased on the type of cell the peptide is expressed in and the desiredremodeled glycan structure. FIG. 52I is a diagram depicting monoclonalantibody to glycoprotein IIb/IIIa-mucin fusion peptides and monoclonalantibody to glycoprotein IIb/IIIa peptides indicating the residues whichbind to glycans contemplated for remodeling, and exemplary glycanformulas linked thereto. FIGS. 52J to 52L are diagrams of contemplatedremodeling steps based on the type of cell the peptide is expressed inand the desired remodeled glycan structure.

[0200]FIG. 53, comprising FIGS. 53A to 53G, sets forth exemplary schemesfor remodeling glycan structures on a monoclonal antibody to CD20(Rituxan™). FIG. 53A is a diagram depicting monoclonal antibody to CD20which have been mutated to contain an N-glycosylation site(s) indicatingthe residue which is linked to glycans contemplated for remodeling, andexemplary glycan formulas linked thereto. FIGS. 53B to 53D are diagramsof contemplated remodeling steps of the glycan of the peptides in FIG.53A based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure. FIG. 53E is a diagram depictingmonoclonal antibody to CD20 which has been mutated to contain anN-glycosylation site(s) indicating the residue(s) which is linked toglycans contemplated for remodeling, and exemplary glycan formulaslinked thereto. FIGS. 53F to 53G are diagrams of contemplated remodelingsteps of the glycan of the peptides in FIG. 53E based on the type ofcell the peptide is expressed in and the desired remodeled glycanstructure.

[0201]FIG. 54, comprising FIGS. 54A to 54O, sets forth exemplary schemesfor remodeling glycan structures on anti-thrombin III (AT III). FIG. 54Ais a diagram depicting the anti-thrombin III peptide indicating theamino acid residues to which an N-linked glycan is linked, and anexemplary glycan formula linked thereto. FIGS. 54B to 54G are diagramsof contemplated remodeling steps of the glycan of the peptide in FIG.54A based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure. FIG. 54H is a diagram depicting theanti-thrombin III peptide indicating the amino acid residues to which anN-linked glycan is linked, and an exemplary glycan formula linkedthereto. FIGS. 54I to 54K are diagrams of contemplated remodeling stepsof the glycan of the peptide in FIG. 54H based on the type of cell thepeptide is expressed in and the desired remodeled glycan structure. FIG.54L is a diagram depicting the anti-thrombin III peptide indicating theamino acid residues to which an N-linked glycan is linked, and anexemplary glycan formula linked thereto. FIGS. 54M to 54O are diagramsof contemplated remodeling steps of the glycan of the peptide in FIG.54L based on the type of cell the peptide is expressed in and thedesired remodeled glycan structure.

[0202]FIG. 55, comprising FIGS. 55A to 55J, sets forth exemplary schemesfor remodeling glycan structures on subunits α and β of human ChorionicGonadotropin (hCG). FIG. 55A is a diagram depicting the hCGα and hCGβpeptides indicating the residues which bind to N-linked glycans (A) andO-linked glycans (B) contemplated for remodeling, and formulas for theglycans. FIGS. 55B to 55J are diagrams of contemplated remodeling stepsbased on the type of cell the peptide is expressed in and the desiredremodeled glycan structure.

[0203]FIG. 56, comprising FIGS. 56A to 56J, sets forth exemplary schemesfor remodeling glycan structures on alpha-galactosidase (Fabrazyme™).FIG. 56A is a diagram depicting the alpha-galactosidase A peptideindicating the amino acid residues which bind to N-linked glycans (A)contemplated for remodeling, and formulas for the glycans. FIGS. 56B to56J are diagrams of contemplated remodeling steps based on the type ofcell the peptide is expressed in and the desired remodeled glycanstructure.

[0204]FIG. 57, comprising FIGS. 57A to 57J, sets forth exemplary schemesfor remodeling glycan structures on alpha-iduronidase (Aldurazyme™).FIG. 57A is a diagram depicting the alpha-iduronidase peptide indicatingthe amino acid residues which bind to N-linked glycans (A) contemplatedfor remodeling, and formulas for the glycans. FIGS. 57B to 57J arediagrams of contemplated remodeling steps based on the type of cell thepeptide is expressed in and the desired remodeled glycan structure.

[0205]FIG. 58, comprising FIGS. 58A and 58B, is an exemplary nucleotideand corresponding amino acid sequence of granulocyte colony stimulatingfactor (G-CSF) (SEQ ID NOS: 1 and 2, respectively).

[0206]FIG. 59, comprising FIGS. 59A and 59B, is an exemplary nucleotideand corresponding amino acid sequence of interferon alpha (IFN-alpha)(SEQ ID NOS: 3 and 4, respectively).

[0207]FIG. 60, comprising FIGS. 60A and 60B, is an exemplary nucleotideand corresponding amino acid sequence of interferon beta (IFN-beta) (SEQID NOS: 5 and 6, respectively).

[0208]FIG. 61, comprising FIGS. 61A and 61B, is an exemplary nucleotideand corresponding amino acid sequence of Factor VIIa (SEQ ID NOS: 7 and8, respectively).

[0209]FIG. 62, comprising FIGS. 62A and 62B, is an exemplary nucleotideand corresponding amino acid sequence of Factor IX (SEQ ID NOS: 9 and10, respectively).

[0210]FIG. 63, comprising FIGS. 63A through 63D, is an exemplarynucleotide and corresponding amino acid sequence of the alpha and betachains of follicle stimulating hormone (FSH), respectively (SEQ ID NOS:11 through 14, respectively).

[0211]FIG. 64, comprising FIGS. 64A and 64B, is an exemplary nucleotideand corresponding amino acid sequence of erythropoietin (EPO) (SEQ IDNOS: 15 and 16, respectively).

[0212]FIG. 65 is an amino acid sequence of mature EPO, i.e. 165 aminoacids (SEQ ID NO:73).

[0213]FIG. 66, comprising FIGS. 66A and 66B, is an exemplary nucleotideand corresponding amino acid sequence of granulocyte-macrophage colonystimulating factor (GM-CSF) (SEQ ID NOS: 17 and 18, respectively).

[0214]FIG. 67, comprising FIGS. 67A and 67B, is an exemplary nucleotideand corresponding amino acid sequence of interferon gamma (IFN-gamma)(SEQ ID NOS: 19 and 20, respectively).

[0215]FIG. 68, comprising FIGS. 68A and 68B, is an exemplary nucleotideand corresponding amino acid sequence of α-1-protease inhibitor (A-1-PI,or α-antitrypsin) (SEQ ID NOS: 21 and 22, respectively).

[0216]FIG. 69, comprising FIGS. 69A-1 to 69A-2, and 69B, is an exemplarynucleotide and corresponding amino acid sequence of glucocerebrosidase(SEQ ID NOS: 23 and 24, respectively).

[0217]FIG. 70, comprising FIGS. 70A and 70B, is an exemplary nucleotideand corresponding amino acid sequence of tissue-type plasminogenactivator (TPA) (SEQ ID NOS: 25 and 26, respectively).

[0218]FIG. 71, comprising FIGS. 71A and 71B, is an exemplary nucleotideand corresponding amino acid sequence of Interleukin-2 (IL-2) (SEQ IDNOS: 27 and 28, respectively).

[0219]FIG. 72, comprising FIGS. 72A-1 through 72A-4 and FIGS. 72B-1through 72B-4, is an exemplary nucleotide and corresponding amino acidsequence of Factor VIII (SEQ ID NOS: 29 and 30, respectively).

[0220]FIG. 73, comprising FIGS. 73A and 73B, is an exemplary nucleotideand corresponding amino acid sequence of urokinase (SEQ ID NOS: 33 and34, respectively).

[0221]FIG. 74, comprising FIGS. 74A and 74B, is an exemplary nucleotideand corresponding amino acid sequence of human recombinant DNase(hrDNase) (SEQ ID NOS: 39 and 40, respectively).

[0222]FIG. 75, comprising FIGS. 75A and 75B, is an exemplary nucleotideand corresponding amino acid sequence of an insulin molecule (SEQ IDNOS: 43 and 44, respectively).

[0223]FIG. 76, comprising FIGS. 76A and 76B, is an exemplary nucleotideand corresponding amino acid sequence of S-protein from a Hepatitis Bvirus (HbsAg) (SEQ ID NOS: 45 and 46, respectively).

[0224]FIG. 77, comprising FIGS. 77A and 77B, is an exemplary nucleotideand corresponding amino acid sequence of human growth hormone (hGH) (SEQID NOS: 47 and 48, respectively).

[0225]FIG. 78, comprising FIGS. 78A and 78D, are exemplary nucleotideand corresponding amino acid sequences of anti-thrombin III. FIGS. 78Aand 78B, are an exemplary nucleotide and corresponding amino acidsequences of “WT” anti-thrombin III (SEQ ID NOS: 63 and 64,respectively).

[0226]FIG. 79, comprising FIGS. 79A to 79D, are exemplary nucleotide andcorresponding amino acid sequences of human chorionic gonadotropin (hCG)α and β subunits. FIGS. 79A and 79B are an exemplary nucleotide andcorresponding amino acid sequence of the α-subunit of human chorionicgonadotropin (SEQ ID NOS: 69 and 70, respectively). FIGS. 79C and 79Dare an exemplary nucleotide and corresponding amino acid sequence of thebeta subunit of human chorionic gonadotrophin (SEQ ID NOS: 71 and 72,respectively).

[0227]FIG. 80, comprising FIGS. 80A and 80B, is an exemplary nucleotideand corresponding amino acid sequence of α-iduronidase (SEQ ID NOS: 65and 66, respectively).

[0228]FIG. 81, comprising FIGS. 81A and 81B, is an exemplary nucleotideand corresponding amino acid sequence of α-galactosidase A (SEQ ID NOS:67 and 68, respectively).

[0229]FIG. 82, comprising FIGS. 82A and 82B, is an exemplary nucleotideand corresponding amino acid sequence of the 75 kDa tumor necrosisfactor receptor (TNF-R), which comprises a portion of Enbrel™ (tumornecrosis factor receptor (TNF-R)/IgG fusion) (SEQ ID NOS: 31 and 32,respectively).

[0230]FIG. 83, comprising FIGS. 83A and 83B, is an exemplary amino acidsequence of the light and heavy chains, respectively, of Herceptin™(monoclonal antibody (MAb) to Her-2, human epidermal growth factorreceptor) (SEQ ID NOS: 35 and 36, respectively).

[0231]FIG. 84, comprising FIGS. 84A and 84B, is an exemplary amino acidsequence the heavy and light chains, respectively, of Synagis™ (MAb to Fpeptide of Respiratory Syncytial Virus) (SEQ ID NOS: 37 and 38,respectively).

[0232]FIG. 85, comprising FIGS. 85A and 85B, is an exemplary nucleotideand corresponding amino acid sequence of the non-human variable regionsof Remicade™ (MAb to TNFα) (SEQ ID NOS: 41 and 42, respectively).

[0233]FIG. 86, comprising FIGS. 86A and 86B, is an exemplary nucleotideand corresponding amino acid sequence of the Fe portion of human IgG(SEQ ID NOS: 49 and 50, respectively).

[0234]FIG. 87 is an exemplary amino acid sequence of the mature variableregion light chain of an anti-glycoprotein IIb/IIIa murine antibody (SEQID NO: 52).

[0235]FIG. 88 is an exemplary amino acid sequence of the mature variableregion heavy chain of an anti-glycoprotein IIb/IIIa murine antibody (SEQID NO: 54).

[0236]FIG. 89 is an exemplary amino acid sequence of variable regionlight chain of a human IgG (SEQ ID NO: 51).

[0237]FIG. 90 is an exemplary amino acid sequence of variable regionheavy chain of a human IgG (SEQ ID NO:53).

[0238]FIG. 91 is an exemplary amino acid sequence of a light chain of ahuman IgG (SEQ ID NO:55).

[0239]FIG. 92 is an exemplary amino acid sequence of a heavy chain of ahuman IgG (SEQ ID NO:56).

[0240]FIG. 93, comprising FIGS. 93A and 93B, is an exemplary nucleotideand corresponding amino acid sequence of the mature variable region ofthe light chain of an anti-CD20 murine antibody (SEQ ID NOS: 59 and 60,respectively).

[0241]FIG. 94, comprising FIGS. 94A and 94B, is an exemplary nucleotideand corresponding amino acid sequence of the mature variable region ofthe heavy chain of an anti-CD20 murine antibody (SEQ ID NOS: 61 and 62,respectively).

[0242]FIG. 95, comprising FIGS. 95A through 95E, is the nucleotidesequence of the tandem chimeric antibody expression vector TCAE 8 (SEQID NO:57).

[0243]FIG. 96, comprising FIGS. 96A through 96E, is the nucleotidesequence of the tandem chimeric antibody expression vector TCAE 8containing the light and heavy variable domains of the anti-CD20 murineantibody (SEQ ID NO:58).

[0244]FIG. 97, comprising FIGS. 97A to 97C, are graphs depicting 2-AAHPLC analysis of glycans released by PNGaseF from myeloma-expressedCri-IgG1 antibody. The structure of the glycans is determined byretention time: the G0 glycoform elutes at 30 min., the G1 glycoformelutes at ˜33 min., the G2 glycoform elutes at about approximately 37min. and the S1-G1 glycoform elutes at ˜70 min. FIG. 97A depicts theanalysis of the DEAE antibody sample. FIG. 97B depicts the analysis ofthe SPA antibody sample. FIG. 97C depicts the analysis of the Fcantibody sample. The percent area under the peaks for these graphs issummarized in Table 14.

[0245]FIG. 98, comprising FIGS. 98A to 98C, are graphs depicting theMALDI analysis of glycans released by PNGaseF from myeloma-expressedCri-IgG1 antibody. The glycans were derivatized with 2-AA and thenanalyzed by MALDI. FIG. 98A depicts the analysis of the DEAE antibodysample. FIG. 98B depicts the analysis of the SPA antibody sample. FIG.98C depicts the analysis of the Fe antibody sample.

[0246]FIG. 99, comprising FIGS. 99A to 99D, are graphs depicting thecapillary electrophoresis analysis of glycans released from Cri-IgG1antibodies that have been glycoremodeled to contain M3N2 glycoforms. Agraph depicting the capillary electrophoresis analysis of glycanstandards derivatized with APTS is shown in FIG. 99A. FIG. 99B depictsthe analysis of the DEAE antibody sample. FIG. 99C depicts the analysisof the SPA antibody sample. FIG. 99D depicts the analysis of the Fcantibody sample. The percent area under the peaks for these graphs issummarized in Table 15.

[0247]FIG. 100, comprising FIGS. 100A to 100D, are graphs depicting thecapillary electrophoresis analysis of glycans released from Cri-IgG1antibodies that have been glycoremodeled to contain G0 glycoforms. Agraph depicting the capillary electrophoresis analysis of glycanstandards derivatized with APTS is shown in FIG. 100A. FIG. 100B depictsthe analysis of the DEAE antibody sample. FIG. 100C depicts the analysisof the SPA antibody sample. FIG. 100D depicts the analysis of the Fcantibody sample. The percent area under the peaks for these graphs issummarized in Table 16.

[0248]FIG. 101, comprising FIGS. 101A to 101C, are graphs depicting 2-AAHPLC analysis of glycans released from Cri-IgG1 antibodies that havebeen glycoremodeled to contain G0 glycoforms. The released glycans werelabeled with 2AA and separated by HPLC on a NH2P-50 4D amino column.FIG. 101A depicts the analysis of the DEAE antibody sample. FIG. 10IBdepicts the analysis of the SPA antibody sample. FIG. 101C depicts theanalysis of the Fc antibody sample. The percent area under the peaks forthese graphs is summarized in Table 16

[0249]FIG. 102, comprising FIGS. 102A to 102C, are graphs depicting theMALDI analysis of glycans released from Cri-IgG1 antibodies that havebeen glycoremodeled to contain G0 glycoforms. The released glycans werederivatized with 2-AA and then analyzed by MALDI. FIG. 102A depicts theanalysis of the DEAE antibody sample. FIG. 102B depicts the analysis ofthe SPA antibody sample. FIG. 102C depicts the analysis of the Fcantibody sample.

[0250]FIG. 103, comprising FIGS. 103A to 103D, are graphs depicting thecapillary electrophoresis analysis of glycans released from Cri-IgG1antibodies that have been glycoremodeled to contain G2 glycoforms. Agraph depicting the capillary electrophoresis analysis of glycanstandards derivatized with APTS is shown in FIG. 103A. FIG. 103B depictsthe analysis of the DEAE antibody sample. FIG. 103C depicts the analysisof the SPA antibody sample. FIG. 103D depicts the analysis of the Fcantibody sample. The percent area under the peaks for these graphs issummarized in Table 17.

[0251]FIG. 104, comprising FIGS. 104A to 104C, are graphs depicting the2-AA HPLC analysis of glycans released from remodeled Cri-IgG1antibodies that have been glycoremodeled to contain G2 glycoforms. Thereleased glycans were labeled with 2AA and then separated by HPLC on aNH2P-50 4D amino column. FIG. 104A depicts the analysis of the DEAEantibody sample. FIG. 104B depicts the analysis of the SPA antibodysample. FIG. 104C depicts the analysis of the Fc antibody sample. Thepercent area under the peaks for these graphs is summarized in Table 17.

[0252]FIG. 105, comprising FIGS. 105A to 105C, are graphs depictingMALDI analysis of glycans released from Cri-IgG1 antibodies that havebeen glycoremodeled to contain G2 glycoforms. The released glycans werederivatized with 2-AA and then analyzed by MALDI. FIG. 105A depicts theanalysis of the DEAE antibody sample. FIG. 105B depicts the analysis ofthe SPA antibody sample. FIG. 105C depicts the analysis of the Fcantibody sample.

[0253]FIG. 106, comprising FIGS. 106A to 106D, are graphs depictingcapillary electrophoresis analysis of glycans released from Cri-IgG1antibodies that have been glycoremodeled by GnT-I treatment of M3N2glycoforms. A graph depicting the capillary electrophoresis analysis ofglycan standards derivatized with APTS is shown in FIG. 106A. FIG. 106Bdepicts the analysis of the DEAE antibody sample. FIG. 106C depicts theanalysis of the SPA antibody sample. FIG. 106D depicts the analysis ofthe Fc antibody sample.

[0254]FIG. 107, comprising FIGS. 107A to 107C, are graphs depicting 2-AAHPLC analysis of glycans released from Cri-IgG1 antibodies that havebeen remodeled by GnT-I treatment of M3N2 glycoforms. The releasedglycans were labeled with 2-AA and separated by HPLC on a NH2P-50 4Damino column. FIG. 107A depicts the analysis of the DEAE antibodysample. FIG. 107B depicts the analysis of the SPA antibody sample. FIG.107C depicts the analysis of the Fc antibody sample.

[0255]FIG. 108, comprising FIGS. 108A to 108C, are graphs depictingMALDI analysis of glycans released from Cri-IgG1 antibodies that havebeen glycoremodeled by GnT-I treatment of M3N2 glycoforms. The releasedglycans were derivatized with 2-AA and then analyzed by MALDI. FIG. 108Adepicts the analysis of the DEAE antibody sample. FIG. 108B depicts theanalysis of the SPA antibody sample. FIG. 108C depicts the analysis ofthe Fc antibody sample.

[0256]FIG. 109, comprising FIGS. 109A to 109D, are graphs depictingcapillary electrophoresis of glycans released from Cri-IgG1 antibodiesthat have been glycoremodeled by GnT-I, II and III treatment of M3N2glycoforms. A graph depicting the capillary electrophoresis analysis ofglycan standards derivatized with APTS is shown in FIG. 109A. FIG. 109Bdepicts the analysis of the DEAE antibody sample. FIG. 109C depicts theanalysis of the SPA antibody sample. FIG. 109D depicts the analysis ofthe Fc antibody sample. The percent area under the peaks for thesegraphs is summarized in Table 18.

[0257]FIG. 110, comprising FIGS. 110A to 110C, are graphs depicting 2-AAHPLC analysis of glycans released from Cri-IgG1 antibodies that havebeen glycoremodeled by GnT-I, II and III treatment of M3N2 glycoforms.The released glycans were labeled with 2AA and then separated by HPLC ona NH2P-50 4D amino column. FIG. 110A depicts the analysis of the DEAEantibody sample. FIG. 110B depicts the analysis of the SPA antibodysample. FIG. 110C depicts the analysis of the Fc antibody sample. Thepercent area under the peaks for these graphs is summarized in Table 18.

[0258]FIG. 111, comprising FIGS. 111A to 111C, are graphs depictingMALDI analysis of glycans released from Cri-IgG1 antibodies that havebeen glycoremodeled by galactosyltransferase treatment of NGA2Fglycoforms. The released glycans were derivatized with 2-AA and thenanalyzed by MALDI. FIG. 111A depicts the analysis of the DEAE antibodysample. FIG. 111B depicts the analysis of the SPA antibody sample. FIG.111C depicts the analysis of the Fc antibody sample.

[0259]FIG. 112, comprising 112A to 112D, are graphs depicting 2-AA HPLCanalysis of glycans released from Cri-IgG1 antibodies containing NGA2Fisoforms before GalT1 treatment (FIGS. 112A and 112C) and after GalT1treatment (FIGS. 112B and 112D). FIGS. 112A and 112B depict the analysisof the DEAE sample of antibodies. FIGS. 112C and 112D depict theanalysis of the Fc sample of antibodies. The released glycans werelabeled with 2AA and separated by HPLC on a NH2P-50 4D amino column.

[0260]FIG. 113, comprising 113A to 113C, are graphs depicting 2-AA HPLCanalysis of glycans released from Cri-IgG1 antibodies that have beenglycoremodeled by ST3Gal3 treatment of G2 glycoforms. The releasedglycans are labeled with 2-AA and then separated by HPLC on a NH2P-50 4Damino column. FIG. 113A depicts the analysis of the DEAE antibodysample. FIG. 113B depicts the analysis of the SPA antibody sample. FIG.113C depicts the analysis of the Fc antibody sample. The percent areaunder the peaks for these graphs is summarized in Table 19.

[0261]FIG. 114, comprising FIGS. 114A to 114C, are graphs depictingMALDI analysis of glycans released from Cri-IgG1 antibodies that hadbeen glycoremodeled by ST3Gal3 treatment of G2 glycoforms. The releasedglycans were derivatized with 2-AA and then analyzed by MALDI. FIG. 114Adepicts the analysis of the DEAE antibody sample. FIG. 114B depicts theanalysis of the SPA antibody sample. FIG. 114C depicts the analysis ofthe Fc antibody sample.

[0262]FIG. 115, comprising FIGS. 115A to 115D, are graphs depictingcapillary electrophoresis analysis of glycans released from Cri-IgG1antibodies that had been glycoremodeled by ST6Gal1 treatment of G2glycoforms. A graph depicting the capillary electrophoresis analysis ofglycan standards derivatized with APTS is shown in FIG. 115A. FIG. 115Bdepicts the analysis of the DEAE antibody sample. FIG. 115C depicts theanalysis of the SPA antibody sample. FIG. 115D depicts the analysis ofthe Fe antibody sample.

[0263]FIG. 116, comprising FIGS. 116A to 116C, are graphs depicting 2-AAHPLC analysis of glycans released from Cri-IgG1 antibodies that had beenglycoremodeled by ST6Gal1 treatment of G2 glycoforms. The releasedglycans were labeled with 2-AA and separated by HPLC on a NH2P-50 4Damino column. FIG. 116A depicts the analysis of the DEAE antibodysample. FIG. 116B depicts the analysis of the SPA antibody sample. FIG.116C depicts the analysis of the Fe antibody sample.

[0264]FIG. 117, comprising FIGS. 117A to 117C, are graphs depictingMALDI analysis of glycans released from Cri-IgG1 antibodies that hadbeen glycoremodeled by ST6Gal1 treatment of G2 glycoforms. The releasedglycans were derivatized with 2-AA and then analyzed by MALDI. FIG. 117Adepicts the analysis of the DEAE antibody sample. FIG. 117B depicts theanalysis of the SPA antibody sample. FIG. 117C depicts the analysis ofthe Fc antibody sample.

[0265]FIG. 118, comprising FIGS. 118A to 118E, depicts images ofSDS-PAGE analysis of the glycoremodeled of Cri-IgG1 antibodies withdifferent glycoforms under non-reducing conditions. Bovine serum albumin(BSA) was run under reducing conditions as a quantitative standard.Protein molecular weight standards are displayed and their size isindicated in kDa. FIG. 118A depicts SDS-PAGE analysis of the DEAE, SPAand Fc Cri-IgG1 antibodies glycoremodeled to contain G0 and G2glycoforms. FIG. 118B depicts SDS-PAGE analysis of the DEAE, SPA and FcCri-IgG1 antibodies glycoremodeled to contain NGA2F (bisecting) andGnT-I-M3N2 (GnT1) glycoforms. FIG. 118C depicts SDS-PAGE analysis of theDEAE, SPA and Fc Cri-IgG1 antibodies glycoremodeled to contain S2G2(ST6Gal1) glycoforms. FIG. 118D depicts SDS-PAGE analysis of the DEAE,SPA and Fe Cri-IgG1 antibodies glycoremodeled to contain M3N2glycoforms, and BSA. FIG. 118E depicts SDS-PAGE analysis of the DEAE,SPA and Fe Cri-IgG1 antibodies glycoremodeled to contain Gal-NGA2F(Gal-bisecting) glycoforms, and BSA.

[0266]FIG. 119 is an image of an acrylamide gel depicting the results ofFACE analysis of the pre- and post-sialylation of TP10. The BiNA₀species has no sialic acid residues. The BiNA₁ species has one sialicacid residue. The BiNA₂ species has two sialic acid residues.Bi=biantennary; NA=neuraminic acid.

[0267]FIG. 120 is a graph depicting the plasma concentration in μg/mlover time of pre- and post-sialylation TP10 injected into rats.

[0268]FIG. 121 is a graph depicting the area under the plasmaconcentration-time curve (AUC) in μg/hr/ml for pre- and post sialylatedTP1O.

[0269]FIG. 122 is an image of an acrylamide gel depicting the results ofFACE glycan analysis of the pre- and post-fucosylation of TP10 and FACEglycan analysis of CHO cell produced TP-20. The BiNA₂F₂ species has twoneuraminic acid (NA) residues and two fucose residues (F).

[0270]FIG. 123 is a graph depicting the in vitro binding of TP20(sCR1sLe^(X)) glycosylated in vitro (diamonds) and in vivo in Lec11 CHOcells (squares).

[0271]FIG. 124 is a graph depicting the analysis by 2-AA HPLC ofglycoforms from the GlcNAc-ylation of EPO.

[0272]FIG. 125, comprising FIGS. 125A and 125B, are graphs depicting the2-AA HPLC analysis of two lots of EPO to which N-acetylglucosamine wasbeen added. FIG. 125A depicts the analysis of lot A, and FIG. 125Bdepicts the analysis of lot B.

[0273]FIG. 126 is a graph depicting the 2-AA HPLC analysis of theproducts the reaction introducing a third glycan branch to EPO withGNT-V.

[0274]FIG. 127 is a graph depicting a MALDI-TOF spectrum of the glycansof the EPO preparation after treatment with GnT-I, GnT-II, GnT-III,GNT-V and GalT1, with appropriate donor groups.

[0275]FIG. 128 is a graph depicting a MALDI spectrum the glycans ofnative EPO.

[0276]FIG. 129 is an image of an SDS-PAGE gel of the products of thePEGylation reactions using CMP-SA-PEG (1 kDa), and CMP-SA-PEG (10 kDa).

[0277]FIG. 130 is a graph depicting the results of the in vitro bioassayof PEGylated EPO. Diamonds represent the data from sialylated EPO havingno PEG molecules. Squares represent the data obtained using EPO with PEG(1 kDa). Triangles represent the data obtained using EPO with PEG (10kDa).

[0278]FIG. 131 is a diagram of CHO-expressed EPO. The EPO polypeptide is165 amino acids in length, with a molecular weight of 18 kDa withoutglycosylation. The glycosylated forms of EPO produced in CHO cells havea molecular weight of about 33 kDa to 39 kDa. The shapes which representthe sugars in the glycan chains are identified in the box at the loweredge of the drawing.

[0279]FIG. 132 is a diagram of insect cell expressed EPO. The shapesthat represent the sugars in the glycan chains are identified in the boxat the lower edge of FIG. 131.

[0280]FIG. 133 is a bar graph depicting the molecular weights of the EPOpeptides expressed in insect cells which were remodeled to form completemono-, bi- and tri-antennary glycans, with optional glycoPEGylation with1 kDa, 10 kDa or 20 kDa PEG. Epoetin™ is EPO expressed in mammaliancells without further glycan modification or PEGylation. NESP (Aranesp™,Amgen, Thousand Oaks, Calif.) is a form of EPO having 5 N-linked glycansites that is also expressed in mammalian cells without further glycanmodification or PEGylation.

[0281]FIG. 134, comprising FIGS. 134A and 134B, depicts one scheme forthe remodeling and glycoPEGylation of insect cell expressed EPO. FIG.134A depicts the remodeling and glycoPEGylation steps that remodel theinsect expressed glycan to a mono-antennary glycoPEGylated glycan. FIG.134B depicts the remodeled EPO polypeptide having a completedglycoPEGylated mono-antennary glycan at each N-linked glycan site of thepolypeptide. The shapes that represent the sugars in the glycan chainsare identified in the box at the lower edge of FIG. 131, except that thetriangle represents sialic acid.

[0282]FIG. 135 is a graph depicting the in vitro bioactivities of EPO-SAand EPO-SA-PEG constructs. The in vitro assay measured the proliferationof TF-1 erythroleukemia cells which were maintained for 48 hr inRBMI+FBS 10%+GM-CSF (12 ng/ml) after the EPO construct was added at10.0, 5.0, 2.0, 1.0, 0.5, and 0 μg/ml. Tri-SA refers to EPO constructswhere the glycans are tri-antennary and have SA. Tri-SA 1K PEG refers toEPO constructs where the glycans are tri-antennary and have Gal and arethen glycoPEGylated with SA-PEG 1 kDa. Di-SA 10K PEG refers to EPOconstructs where the glycans are bi-antennary and have Gal and are thenglycoPEGylated with SA-PEG 10 kDa. Di-SA 1K PEG refers to EPO constructswhere the glycans are bi-antennary and have Gal and are thenglycoPEGylated with SA-PEG 1 kDa. Di-SA refers to EPO constructs wherethe glycans are bi-antennary and are built out to SA. Epogen™ is EPOexpressed in CHO cells with no further glycan modification.

[0283]FIG. 136 is a graph depicting the pharmacokinetics of the EPOconstructs in rat. Rats were bolus injected with [I¹²⁵]-labeledglycoPEGylated and non-glycoPEGylated EPO. The graph shows theconcentration of the radio-labeled EPO in the bloodstream of the rat at0 to about 72 minutes after injection. “Biant-10K” refers to EPO withbiantennary glycan structures with terminal 10 kDa PEG moieties.“Mono-20K” refers to EPO with monoantennary glycan structures withterminal 20 kDa PEG moieties. NESP refers to the commercially availableAranesp. “Biant-1K” refers to EPO with biantennary glycan structureswith terminal 1 kDa PEG moieties. “Biant-SA” refers to EPO withbiantennary glycan structures with terminal 1 kDa moieties. Theconcentration of the EPO constructs in the bloodstream at 72 hr. is asfollows: Biant-10K, 5.1 cpm/ml; Mono-20K, 3.2 cpm/ml; NESP, 1 cpm/ml;and Biant-1K, 0.2 cpm/ml; Biant-SA, 0.1 cpm/ml. The relative area underthe curve of the EPO constructs is as follows: Biant-10K, 2.9; Mono-20K,2.1; NESP, 1; Biant-1K, 0.5; and Biant-SA, 0.2.

[0284]FIG. 137 is a bar graph depicting the ability of the EPOconstructs to stimulate reticulocytosis in vivo. Each treatment group iscomposed of eight mice. Mice were given a single subcutaneous injectionof 10 μg protein/kg body weight. The percent reticulocytosis wasmeasured at 96 hr. Tri-antennary-SA2,3(6) construct has the SA moleculebonded in a 2, 3 or 2,6 linkage (see, Example 18 herein for preparation)wherein the glycan on EPO is tri-antennary N-glycans with SA-PEG 10 K isattached thereon. Similarly, bi-antennary-10K PEG is EPO having abi-antennary N-glycan with SA-PEG at 10 K PEG attached thereon.

[0285]FIG. 138 is a bar graph depicting the ability of EPO constructs toincrease the hematocrit of the blood of mice in vivo. CD-1 female micewere injected i.p. with 2.5 μg protein/kg body weight. The hematocrit ofthe mice was measured on day 15 after the EPO injection. Bi-1k refers toEPO constructs where the glycans are bi-antennary and are built out tothe Gal and then glycoPEGylated with SA-PEG 1 kDa. Mono-20k refers toEPO constructs where the glycans are mono-antennary and are built out tothe Gal and then glycoPEGylated with SA-PEG 20 kDa.

[0286]FIG. 139, comprising FIGS. 139A and 139B, depicts the analysis ofglycans enzymatically released from EPO expressed in insect cells(Protein Sciences, Lot # 060302). FIG. 139A depicts the HPLC analysis ofthe released glycans. FIG. 139B depicts the MALDI analysis of thereleased glycans. Diamonds represent fucose, and squares representGlcNAc, circles represent mannose.

[0287]FIG. 140 depicts the MALDI analysis of glycans released from EPOafter the GnT-I/GalT-1 reaction. The structures of the glycans have beendetermined by comparison of the peak spectrum with that of standardglycans. The glycan structures are depicted beside the peaks. Diamondsrepresent fucose, and squares represent GlcNAc, circles representmannose, stars represent galactose.

[0288]FIG. 141 depicts the SDS-PAGE analysis of EPO after theGnT-I/GalT-1 reaction, Superdex 75 purification, ST3Gal3 reaction withSA-PEG (10 kDa) and SA-PEG (20 kDa).

[0289]FIG. 142 depicts the results of the TF-1 cell in vitro bioassay ofPEGylated mono-antennary EPO.

[0290]FIG. 143, comprising FIGS. 143A and 143B, depicts the analysis ofglycan released from EPO after the GnT-I/GnT-II reaction. FIG. 143Adepicts the HPLC analysis of the released glycans, where peak 3represents the bi-antennary GlcNAc glycan. FIG. 143B depicts the MALDIanalysis of the released glycans. The structures of the glycans havebeen determined by comparison of the peak spectrum with that of standardglycans. The glycan structures are depicted beside the peaks. Diamondsrepresent fucose, and squares represent GlcNAc, circles representmannose.

[0291]FIG. 144, comprising FIGS. 144A and 144B, depict the HPLC analysisof glycans released from EPO after the GalT-1 reaction. FIG. 144Adepicts the glycans released after the small scale GalT-1 reaction. FIG.144B depicts the glycans released after the large scale GalT-1 reaction.In both figures, Peak 1 is the bi-antennary glycan with terminalgalactose moieties and Peak 2 is the bi-antennary glycan withoutterminal galactose moieties.

[0292]FIG. 145 depicts the Superdex 75 chromatography separation of EPOspecies after the GalT-1 reaction. Peak 2 contains EPO with bi-antennaryglycans with terminal galactose moieties.

[0293]FIG. 146 depicts the SDS-PAGE analysis of each of the products ofthe glycoremodeling process to make bi-antennary glycans with terminalgalactose moieties.

[0294]FIG. 147 depicts the SDS-PAGE analysis of EPO after ST3Gal3sialylation or PEGylation with SA-PEG (1 kDa) or SA-PEG (10 kDa).

[0295]FIG. 148 depicts the HPLC analysis of glycans released from EPOafter the GnT-I/GnT-II reaction. The structures of the glycans have beendetermined by comparison of the peak retention with that of standardglycans. The glycan structures are depicted beside the peaks. Diamondsrepresent fucose, and squares represent GlcNAc, circles representmannose.

[0296]FIG. 149 depicts the HPLC analysis of glycans released from EPOafter the GnT-V reaction. The structures of the glycans have beendetermined by comparison of the peak retention with that of standardglycans. The glycan structures are depicted beside the peaks. Diamondsrepresent fucose, and squares represent GlcNAc, circles representmannose.

[0297]FIG. 150 depicts the HPLC analysis of glycans released from EPOafter the GalT-1 reaction. The structures of the glycans have beendetermined by comparison of the peak retention with that of standardglycans. The glycan structures are depicted beside the peaks. Diamondsrepresent fucose, and squares represent GlcNAc, circles representmannose, open circles represent galactose and triangles represent sialicacid.

[0298]FIG. 151 depicts the HPLC analysis of glycans released from EPOafter the ST3Gal3 reaction. The structures of the glycans have beendetermined by comparison of the peak retention with that of standardglycans. The glycan structures are depicted beside the peaks. Diamondsrepresent fucose, and squares represent GlcNAc, circles representmannose, open circles represent galactose and triangles represent sialicacid.

[0299]FIG. 152 depicts the HPLC analysis of glycans released from EPOafter the ST6Gal1 reaction. The structures of the glycans have beendetermined by comparison of the peak retention with that of standardglycans. The glycan structures are depicted beside the peaks.

[0300]FIG. 153 depicts the results of the TF-1 cells in vitro bioassayof EPO with bi-antennary and triantennary glycans. “Di-SA” refers to EPOwith bi-antennary glycans that terminate in sialic acid. “Di-SA 10K PEG”refers to EPO with bi-antennary glycans that terminate in sialic acidderivatized with PEG (10 kDa). “Di-SA 1K PEG” refers to EPO withbi-antennary glycans that terminate in sialic acid derivatized with PEG(1 kDa). “Tri-SA ST6+ST3” refers to EPO with tri-antennary glycansterminating in 2,6-SA capped with 2,3-SA. “Tri-SA ST3” refers to EPOwith tri-antennary glycans terminating in 2,3-SA.

[0301]FIG. 154 is an image of an IEF gel depicting the pI of theproducts of the desialylation procedure. Lanes 1 and 5 are IEFstandards. Lane 2 is Factor IX protein. Lane 3 is rFactor IX protein.Lane 4 is the desialylation reaction of rFactor IX protein at 20 hr.

[0302]FIG. 155 is an image of an SDS-PAGE gel depicting the molecularweight of Factor IX conjugated with either SA-PEG (1 kDa) or SA-PEG (10kDa) after reaction with CMIP-SA-PEG. Lanes 1 and 6 are SeeBlue+2molecular weight standards. Lane 2 is rF-IX. Lane 3 is desialylatedrF-IX. Lane 4 is rFactor IX conjugated to SA-PEG (1 kDa). Lane 5 isrFactor IX conjugated to SA-PEG (10 kDa).

[0303]FIG. 156 is an image of an SDS-PAGE gel depicting the reactionproducts of direct-sialylation of Factor-IX and sialic acid capping ofFactor-IX-SA-PEG. Lane 1 is protein standards, lane 2 is blank; lane 3is rFactor-IX; lane 4 is SA capped rFactor-IX-SA-PEG (10 kDa); lane 5 isrFactor-IX-SA-PEG (10 kDa); lane 6 is ST3Gal1; lane 7 is ST3Gal3; lanes8, 9, 10 are rFactor-IX-SA-PEG (10 kDa) with no prior sialidasetreatment.

[0304]FIG. 157 is an image of an isoelectric focusing gel (pH 3-7) ofasialo-Factor VIIa. Lane I is rFactor VIIa; lanes 2-5 are asialo-FactorVIIa.

[0305]FIG. 158 is a graph of a MALDI spectra of Factor VIIa.

[0306]FIG. 159 is a graph of a MALDI spectra of Factor VIIa-PEG (1 kDa).

[0307]FIG. 160 is a graph depicting a MALDI spectra of Factor VIIa-PEG(10 kDa).

[0308]FIG. 161 is an image of an SDS-PAGE gel of PEGylated Factor VIIa.Lane 1 is asialo-Factor VIIa. Lane 2 is the product of the reaction ofasialo-Factor VIIa and CMP-SA-PEG (1 kDa) with ST3Gal3 after 48 hr. Lane3 is the product of the reaction of asialo-Factor VIIa and CMP-SA-PEG (1kDa) with ST3Gal3 after 48 hr. Lane 4 is the product of the reaction ofasialo-Factor VIIa and CMP-SA-PEG (10 kDa) with ST3Gal3 at 96 hr.

[0309]FIG. 162 is an image of an isoelectric focusing (IEF) geldepicting the products of the desialylation reaction of human pituitaryFSH. Lanes 1 and 4 are isoelectric focusing (IEF) standards. Lane 2 isnative FSH. Lane 3 is desialylated FSH.

[0310]FIG. 163 is an image of an SDS-PAGE gel of the products of thereactions to make PEG-sialylation of rFSH. Lanes 1 and 8 are SeeBlue+2molecular weight standards. Lane 2 is 15 μg of native FSH. Lane 3 is 15μg of asialo-FSH (AS-FSH). Lane 4 is 15 μg of the products of thereaction of AS-FSH with CMP-SA. Lane 5 is 15 μg of the products of thereaction of AS-FSH with CMP-SA-PEG (1 kDa). Lane 6 is 15 μg of theproducts of the reaction of AS-FSH with CMP-SA-PEG (5 kDa). Lane 7 is 15μg of the products of the reaction of AS-FSH with CMP-SA-PEG (10 kDa).

[0311]FIG. 164 is an image of an isoelectric focusing gel of theproducts of the reactions to make PEG-sialylation of FSH. Lanes 1 and 8are IEF standards. Lane 2 is 15 μg of native FSH. Lane 3 is 15 μg ofasialo-FSH (AS-FSH). Lane 4 is 15 μg of the products of the reaction ofAS-FSH with CMP-SA. Lane 5 is 15 μg of the products of the reaction ofAS-FSH with CMP-SA-PEG (1 kDa). Lane 6 is 15 μg of the products of thereaction of AS-FSH with CMP-SA-PEG (5 kDa). Lane 7 is 15 μg of theproducts of the reaction of AS-FSH with CMP-SA-PEG (10 kDa).

[0312]FIG. 165 is an image of an SDS-PAGE gel of native non-recombinantFSH produced in human pituitary cells. Lanes 1, 2 and 5 are SeeBlue™+2molecular weight standards. Lanes 3 and 4 are native FSH at 5 μg and 25μg, respectively.

[0313]FIG. 166 is an image of an isoelectric focusing gel (pH 3-7)depicting the products of the asialylation reaction of rFSH. Lanes 1 and4 are IEF standards. Lane 2 is native rFSH. Lane 3 is asialo-rFSH.

[0314]FIG. 167 is an image of an SDS-PAGE gel depicting the results ofthe PEG-sialylation of asialo-rFSH. Lane 1 is native rFSH. Lane 2 isasialo-FSH. Lane 3 is the products of the reaction of asialo-FSH andCMP-SA. Lanes 4-7 are the products of the reaction between asialoFSH and0.5 mM CMP-SA-PEG (10 kDa) at 2 hr, 5 hr, 24 hr, and 48 hr,respectively. Lane 8 is the products of the reaction between asialo-FSHand 1.0 mM CMP-SA-PEG (10 kDa) at 48 hr. Lane 9 is the products of thereaction between asialo-FSH and 1.0 mM CMP-SA-PEG (1 kDa) at 48 hr.

[0315]FIG. 168 is an image of an isoelectric focusing gel showing theproducts of PEG-sialylation of asialo-rFSH with a CMP-SA-PEG (1 kDa).Lane 1 is native rFSH. Lane 2 is asialo-rFSH. Lane 3 is the products ofthe reaction of asialo-rFSH and CMP-SA at 24 hr. Lanes 4-7 are theproducts of the reaction of asialo-rFSH and 0.5 mM CMP-SA-PEG (1 kDa) at2 hr, 5 hr, 24 hr, and 48 hr, respectively. Lane 8 is blank. Lanes 9 and10 are the products of the reaction at 48 hr of asialo-rFSH andCMP-SA-PEG (10 kDa) at 0.5 mM and 1.0 mM, respectively.

[0316]FIG. 169 is graph of the pharmacokinetics of rFSH and rFSH-SA-PEG(1 kDa and 10 kDa). This graph illustrates the relationship between thetime a rFSH compound is in the blood stream of the rat, and the meanconcentration of the rFSH compound in the blood for glycoPEGylated rFSHas compared to non-PEGylated rFSH.

[0317]FIG. 170 is a graph of the results of the FSH bioassay usingSertoli cells. This graph illustrates the relationship between the FSHconcentration in the Sertoli cell incubation medium and the amount of17-β estradiol released from the Sertoli cells.

[0318]FIG. 171 is a graph depicting the results of the Steelman-Pohleybioassay of glycoPEGylated and non-glycoPEGylated FSH. Rats weresubcutaneously injected with human chorionic gonadotropin and varyingamounts of FSH for three days, and the average ovarian weight of thetreatment group determined on day 4. rFSH-SA-PEG refers to recombinantFSH that has been glycoPEGylated with PEG (1 kDa). rFSH refers tonon-glycoPEGylated FSH. Each treatment group contains 10 rats.

[0319]FIG. 172, comprising FIGS. 172A and 172B, depicts the chromatogramof INF-β elution from a Superdex-75 column. FIG. 172A depicts the entirechromatogram. FIG. 172B depicts the boxed area of FIG. 172A containingpeaks 4 and 5 in greater detail.

[0320]FIG. 173, comprising FIGS. 173A and 173B, depict MALDI analysis ofglycans enzymatically released from INF-β. FIG. 173A depicts the MALDIanalysis glycans released from native INF-β. FIG. 173B depicts the MALDIanalysis of glycans released from desialylated INF-β. The structures ofthe glycans have been determined by comparison of the peak spectrum withthat of standard glycans. The glycan structures are depicted beside thepeaks. Squares represent GlcNAc, triangles represent fucose, circlesrepresent mannose, diamonds represent galactose and stars representsialic acid.

[0321]FIG. 174 depicts the lectin blot analysis of the sialylation ofthe desialylated INF-β. The blot on the right side is detected withMaackia amurensis agglutinin (MAA) labeled with digoxogenin (DIG) (RocheApplied Science, Indianapolis, IL) to detect α2,3-sialylation. The bloton the left is detected with Erthrina cristagalli lectin (ECL) labeledwith biotin (Vector Laboratories, Burlingame, Calif.) to detect exposedgalactose residues.

[0322]FIG. 175 depicts the SDS-PAGE analysis of the products of the PEG(10 kDa) PEGylation reaction of INF-β. “-PEG” refers to INF-β before thePEGylation reaction. “+PEG” refers to INF-β after the PEGylationreaction.

[0323]FIG. 176 depicts the SDS-PAGE analysis of the products of the PEG(20 kDa) PEGylation reaction of INF-β. “Unmodified” refers to INF-βbefore the PEGylation reaction. “Pegylated” refers to INF-β after thePEGylation reaction.

[0324]FIG. 177 depicts the chromatogram of PEG (10 kDa) PEGylated INF-βelution from a Superdex-200 column.

[0325]FIG. 178 depicts the results of a bioassay of peak fractions ofPEG (10 kDa) PEGylated INF-β shown in the chromatogram depicted FigureINF-PEG 6.

[0326]FIG. 179 depicts the chromatogram of PEG (20 kDa) PEGylated INF-βelution from a Superdex-200 column.

[0327]FIG. 180, comprising FIGS. 180A and 180B, is two graphs depictingthe MALDI-TOF spectrum of RNaseB (FIG. 180A) and the HPLC profile of theoligosaccharides cleaved from RNaseB by N-Glycanase (FIG. 180B). Themajority of N-glycosylation sites of the peptide are modified with highmannose oligosaccharides consisting of 5 to 9 mannose residues.

[0328]FIG. 181 is a scheme depicting the conversion of high mannoseN-Glycans to hybrid N-Glycans. Enzyme 1 is α1,2-mannosidase, fromTrichodoma reesei or Aspergillus saitoi. Enzyme 2 is GnT-I(β-1,2-N-acetyl glucosaminyl transferase I). Enzyme 3 is GalT-I(β1,4-galactosyltransfease 1). Enzyme 4 is α2,3-sialyltransferase orα2,6-sialyltransferase.

[0329]FIG. 182, comprising FIGS. 182A and 182B, is two graphs depictingthe MALDI-TOF spectrum of RNaseB treated with a recombinant T. reeseiα1,2-mannosidase (FIG. 182A) and the HPLC profile of theoligosaccharides cleaved by N-Glycanase from the modified RNaseB (FIG.182B).

[0330]FIG. 183 is a graph depicting the MALDI-TOF spectrum of RNaseBtreated with a commercially available α1,2-mannosidase purified from A.saitoi (Glyko & CalBioChem).

[0331]FIG. 184 is a graph depicting the MALDI-TOF spectrum of modifiedRNaseB by treating the product shown in FIG. 182 with a recombinantGnT-I (GlcNAc transferase-I).

[0332]FIG. 185 is a graph depicting the MALDI-TOF spectrum of modifiedRNaseB by treating the product shown in FIG. 184 with a recombinant GalT1 (galactosyltransferase 1).

[0333]FIG. 186 is a graph depicting the MALDI-TOF spectrum of modifiedRNaseB by treating the product shown in FIG. 185 with a recombinantST3Gal III α2,3-sialyltransferase III) using CMP-SA as the donor for thetransferase.

[0334]FIG. 187 is a graph depicting the MALDI-TOF spectrum of modifiedRNaseB by treating the product shown in FIG. 185 with a recombinantST3Gal III α2,3-sialyltransferase III) using CMP-SA-PEG (10 kDa) as thedonor for the transferase.

[0335]FIG. 188 is a series of schemes depicting the conversion of highmannose N-glycans to complex N-glycans. Enzyme 1 is α1,2-mannosidasefrom Trichoderma reesei or Aspergillus saitoi. Enzyme 2 is GnT-I. Enzyme3 is GalT 1. Enzyme 4 is α2,3-sialyltransferase orα2,6-sialyltransferase. Enzyme 5 is α-mannosidase II. Enzyme 6 isα-mannosidase. Enzyme 7 is GnT-II. Enzyme 8 is α1,6-mannosidase. Enzyme9 is α1,3-mannosidase.

[0336]FIG. 189 is a diagram of the linkage catalyzed byN-acetylglucosaminyltransferase I to VI (GnT I-VI).R=GlcNAβ1,4GlcNAc-Asn-X.

[0337]FIG. 190 is an image of an SDS-PAGE gel: standard (Lane 1); nativetransferrin (Lane 2); asialotransferrin (Lane 3); asialotransferrin andCMP-SA (Lane 4); Lanes 5 and 6, asialotransferrin and CMP-SA-PEG (1 kDa)at 0.5 mM and 5 mM, respectively; Lanes 7 and 8, asialotransferrin andCMP-SA-PEG (5 kDa) at 0.5 mM and 5 mM, respectively; Lanes 9 and 10,asialotransferrin and CMP-SA-PEG (10 kDa) at 0.5 mM and 5 mM,respectively.

[0338]FIG. 191 is an image of an IEF gel: native transferrin (Lane 1);asialotransferrin (Lane 2); asialotransferrin and CMP-SA, 24 hr (Lane3); asialotransferrin and CMP-SA, 96 hr (Lane 4) Lanes 5 and 6,asialotransferrin and CMP-SA-PEG (1 kDa) at 24 hr and 96 hr,respectively; Lanes 7 and 8, asialotransferrin and CMP-SA-PEG (5 kDa) at24 hr and 96 hr, respectively; Lanes 9 and 10, asialotransferrin andCMP-SA-PEG (10 kDa) at 24 hr and 96 hr, respectively.

DETAILED DESCRIPTION OF THE INVENTION

[0339] The present invention includes methods and compositions for thecell free in vitro addition and/or deletion of sugars to or from apeptide molecule in such a manner as to provide a glycopeptide moleculehaving a specific customized or desired glycosylation pattern, whereinthe glycopeptide is produced at an industrial scale. In a preferredembodiment of the invention, the glycopeptide so produced has attachedthereto a modified sugar that has been added to the peptide via anenzymatic reaction. A key feature of the invention is to take a peptideproduced by any cell type and generate a core glycan structure on thepeptide, following which the glycan structure is then remodeled in vitroto generate a glycopeptide having a glycosylation pattern suitable fortherapeutic use in a mammal. More specifically, it is possible accordingto the present invention, to prepare a glycopeptide molecule having amodified sugar molecule or other compound conjugated thereto, such thatthe conjugated molecule confers a beneficial property on the peptide.According to the present invention, the conjugate molecule is added tothe peptide enzymatically because enzyme-based addition of conjugatemolecules to peptides has the advantage of regioselectivity andstereoselectivity. The glycoconjugate may be added to the glycan on apeptide before or after glycosylation has been completed. In otherwords, the order of glycosylation with respect to glycoconjugation maybe varied as described elsewhere herein. It is therefore possible, usingthe methods and compositions provided herein, to remodel a peptide toconfer upon the peptide a desired glycan structure preferably having amodified sugar attached thereto. It is also possible, using the methodsand compositions of the invention to generate peptide molecules havingdesired and or modified glycan structures at an industrial scale,thereby, for the first time, providing the art with a practical solutionfor the efficient production of improved therapeutic peptides.

[0340] Definitions

[0341] Unless defined otherwise, all technical and scientific terms usedherein generally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, and nucleic acidchemistry and hybridization are those well known and commonly employedin the art. Standard techniques are used for nucleic acid and peptidesynthesis. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (e.g., Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, 2d ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.), which are provided throughout this document. The nomenclatureused herein and the laboratory procedures used in analytical chemistryand organic syntheses described below are those well known and commonlyemployed in the art. Standard techniques or modifications thereof, areused for chemical syntheses and chemical analyses.

[0342] The articles “a” and “an” are used herein to refer to one or tomore than one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

[0343] The term “antibody,” as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. Antibodies are typically tetramers ofimmunoglobulin molecules. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, Fv, Fab and F(ab)₂, as well as singlechain antibodies and humanized antibodies (Harlow et al., 1999, UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold SpringHarbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0344] By the term “synthetic antibody” as used herein, is meant anantibody which is generated using recombinant DNA technology, such as,for example, an antibody expressed by a bacteriophage as describedherein. The term should also be construed to mean an antibody which hasbeen generated by the synthesis of a DNA molecule encoding the antibodyand which DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

[0345] As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property by whichit is characterized. A functional enzyme, for example, is one whichexhibits the characteristic catalytic activity by which the enzyme ischaracterized.

[0346] As used herein, the structure

[0347] , is the point of connection between an amino acid or an aminoacid sidechain in the peptide chain and the glycan structure.

[0348] “N-linked” oligosaccharides are those oligosaccharides that arelinked to a peptide backbone through asparagine, by way of anasparagine-N-acetylglucosamine linkage. N-linked oligosaccharides arealso called “N-glycans.” All N-linked oligosaccharides have a commonpentasaccharide core of Man₃GlcNAc₂. They differ in the presence of, andin the number of branches (also called antennae) of peripheral sugarssuch as N-acetylglucosamine, galactose, N-acetylgalactosamine, fucoseand sialic acid. Optionally, this structure may also contain a corefucose molecule and/or a xylose molecule.

[0349] An “elemental trimannosyl core structure” refers to a glycanmoiety comprising solely a trimannosyl core structure, with noadditional sugars attached thereto. When the term “elemental” is notincluded in the description of the “trimannosyl core structure,” thenthe glycan comprises the trimannosyl core structure with additionalsugars attached thereto. Optionally, this structure may also contain acore fucose molecule and/or a xylose molecule.

[0350] The term “elemental trimannosyl core glycopeptide” is used hereinto refer to a glycopeptide having glycan structures comprised primarilyof an elemental trimannosyl core structure. Optionally, this structuremay also contain a core fucose molecule and/or a xylose molecule.

[0351] “O-linked” oligosaccharides are those oligosaccharides that arelinked to a peptide backbone through threonine, serine, hydroxyproline,tyrosine, or other hydroxy-containing amino acids.

[0352] All oligosaccharides described herein are described with the nameor abbreviation for the non-reducing saccharide (i.e., Gal), followed bythe configuration of the glycosidic bond (α or β), the ring bond (1 or2), the ring position of the reducing saccharide involved in the bond(2, 3, 4, 6 or 8), and then the name or abbreviation of the reducingsaccharide (i.e., GlcNAc). Each saccharide is preferably a pyranose. Fora review of standard glycobiology nomenclature see, Essentials ofGlycobiology Varki et al. eds., 1999, CSHL Press.

[0353] The term “sialic acid” refers to any member of a family ofnine-carbon carboxylated sugars. The most common member of the sialicacid family is N-acetyl-neuraminic acid(2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onicacid (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member ofthe family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which theN-acetyl group of NeuAc is hydroxylated. A third sialic acid familymember is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. (1986) J.Biol. Chem. 261: 11550-11557; Kanamori et al., J. Biol. Chem. 265:21811-21819 (1990)). Also included are 9-substituted sialic acids suchas a 9-O—C₁-C₆ acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac,9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of thesialic acid family, see, e.g., Varki, Glycobiology 2: 25-40 (1992);Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed.(Springer-Verlag, New York (1992)). The synthesis and use of sialic acidcompounds in a sialylation procedure is disclosed in internationalapplication WO 92/16640, published Oct. 1, 1992.

[0354] A peptide having “desired glycosylation”, as used herein, is apeptide that comprises one or more oligosaccharide molecules which arerequired for efficient biological activity of the peptide.

[0355] A “disease” is a state of health of an animal wherein the animalcannot maintain homeostasis, and wherein if the disease is notameliorated then the animal's health continues to deteriorate.

[0356] The “area under the curve” or “AUC”, as used herein in thecontext of administering a peptide drug to a patient, is defined astotal area under the curve that describes the concentration of drug insystemic circulation in the patient as a function of time from zero toinfinity.

[0357] The term “half-life” or “t ½”, as used herein in the context ofadministering a peptide drug to a patient, is defined as the timerequired for plasma concentration of a drug in a patient to be reducedby one half. There may be more than one half-life associated with thepeptide drug depending on multiple clearance mechanisms, redistribution,and other mechanisms well known in the art. Usually, alpha and betahalf-lives are defined such that the alpha phase is associated withredistribution, and the beta phase is associated with clearance.However, with protein drugs that are, for the most part, confined to thebloodstream, there can be at least two clearance half-lives. For someglycosylated peptides, rapid beta phase clearance may be mediated viareceptors on macrophages, or endothelial cells that recognize terminalgalactose, N-acetylgalactosamine, N-acetylglucosamine, mannose, orfucose. Slower beta phase clearance may occur via renal glomerularfiltration for molecules with an effective radius <2 nm (approximately68 kD) and/or specific or non-specific uptake and metabolism in tissues.GlycoPEGylation may cap terminal sugars (e.g. galactose orN-acetylgalactosamine) and thereby block rapid alpha phase clearance viareceptors that recognize these sugars. It may also confer a largereffective radius and thereby decrease the volume of distribution andtissue uptake, thereby prolonging the late beta phase. Thus, the preciseimpact of glycoPEGylation on alpha phase and beta phase half-lives willvary depending upon the size, state of glycosylation, and otherparameters, as is well known in the art. Further explanation of“half-life” is found in Pharmaceutical Biotechnology (1997, DFACrommelin and RD Sindelar, eds., Harwood Publishers, Amsterdam, pp101-120).

[0358] The term “residence time”, as used herein in the context ofadministering a peptide drug to a patient, is defined as the averagetime that drug stays in the body of the patient after dosing.

[0359] An “isolated nucleic acid” refers to a nucleic acid segment orfragment which has been separated from sequences which flank it in anaturally occurring state, e.g., a DNA fragment which has been removedfrom the sequences which are normally adjacent to the fragment, e.g.,the sequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid nucleic acid encodingadditional peptide sequence.

[0360] A “polynucleotide” means a single strand or parallel andanti-parallel strands of a nucleic acid. Thus, a polynucleotide may beeither a single-stranded or a double-stranded nucleic acid.

[0361] The term “nucleic acid” typically refers to largepolynucleotides. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides.

[0362] Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction. Thedirection of 5′ to 3′ addition of nucleotides to nascent RNA transcriptsis referred to as the transcription direction. The DNA strand having thesame sequence as an mRNA is referred to as the “coding strand”;sequences on the DNA strand which are located 5′ to a reference point onthe DNA are referred to as “upstream sequences”; sequences on the DNAstrand which are 3′ to a reference point on the DNA are referred to as“downstream sequences.”

[0363] “Encoding” refers to the inherent property of specific sequencesof nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA,to serve as templates for synthesis of other polymers and macromoleculesin biological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a nucleic acid sequenceencodes a protein if transcription and translation of mRNA correspondingto that nucleic acid produces the protein in a cell or other biologicalsystem. Both the coding strand, the nucleotide sequence of which isidentical to the mRNA sequence and is usually provided in sequencelistings, and the non-coding strand, used as the template fortranscription of a gene or cDNA, can be referred to as encoding theprotein or other product of that nucleic acid or cDNA.

[0364] Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

[0365] “Homologous” as used herein, refers to the subunit sequencesimilarity between two polymeric molecules, e.g., between two nucleicacid molecules, e.g., two DNA molecules or two RNA molecules, or betweentwo peptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous then the twosequences are 50% homologous, if 90% of the positions, e.g., 9 of 10,are matched or homologous, the two sequences share 90% homology. By wayof example, the DNA sequences 3′ATTGCC5′ and 3′TATGGC share 50%homology.

[0366] As used herein, “homology” is used synonymously with “identity.”

[0367] The determination of percent identity between two nucleotide oramino acid sequences can be accomplished using a mathematical algorithm.For example, a mathematical algorithm useful for comparing two sequencesis the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci.USA 87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl.Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into theNBLAST and XBLAST programs of Altschul, et al. (1990, J. Mol. Biol.215:403-410), and can be accessed, for example at the National Centerfor Biotechnology Information (NCBI) world wide web site having theuniversal resource locator “http://www.ncbi.nim.nih.gov/BLAST/”. BLASTnucleotide searches can be performed with the NBLAST program (designated“blastn” at the NCBI web site), using the following parameters: gappenalty=5; gap extension penalty=2; mismatch penalty=3; match reward=1;expectation value 10.0; and word size=11 to obtain nucleotide sequenceshomologous to a nucleic acid described herein. BLAST protein searchescan be performed with the XBLAST program (designated “blastn” at theNCBI web site) or the NCBI “blastp” program, using the followingparameters: expectation value 10.0, BLOSUM62 scoring matrix to obtainamino acid sequences homologous to a protein molecule described herein.To obtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al. (1997, Nucleic Acids Res.25:3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used toperform an iterated search which detects distant relationships betweenmolecules (Id.) and relationships between molecules which share a commonpattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blastprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0368] The percent identity between two sequences can be determinedusing techniques similar to those described above, with or withoutallowing gaps. In calculating percent identity, typically exact matchesare counted.

[0369] A “heterologous nucleic acid expression unit” encoding a peptideis defined as a nucleic acid having a coding sequence for a peptide ofinterest operably linked to one or more expression control sequencessuch as promoters and/or repressor sequences wherein at least one of thesequences is heterologous, i.e., not normally found in the host cell.

[0370] By describing two polynucleotides as “operably linked” is meantthat a single-stranded or double-stranded nucleic acid moiety comprisesthe two polynucleotides arranged within the nucleic acid moiety in sucha manner that at least one of the two polynucleotides is able to exert aphysiological effect by which it is characterized upon the other. By wayof example, a promoter operably linked to the coding region of a nucleicacid is able to promote transcription of the coding region.

[0371] As used herein, the term “promoter/regulatory sequence” means anucleic acid sequence which is required for expression of a gene productoperably linked to the promoter/regulator sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

[0372] A “constitutive promoter is a promoter which drives expression ofa gene to which it is operably linked, in a constant manner in a cell.By way of example, promoters which drive expression of cellularhousekeeping genes are considered to be constitutive promoters.

[0373] An “inducible” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

[0374] A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

[0375] A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

[0376] “Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses that incorporate the recombinant polynucleotide.

[0377] A “genetically engineered” or “recombinant” cell is a cell havingone or more modifications to the genetic material of the cell. Suchmodifications are seen to include, but are not limited to, insertions ofgenetic material, deletions of genetic material and insertion of geneticmaterial that is extrachromasomal whether such material is stablymaintained or not.

[0378] A “peptide” is an oligopeptide, polypeptide, peptide, protein orglycoprotein. The use of the term “peptide” herein includes a peptidehaving a sugar molecule attached thereto when a sugar molecule isattached thereto.

[0379] As used herein, “native form” means the form of the peptide whenproduced by the cells and/or organisms in which it is found in nature.When the peptide is produced by a plurality of cells and/or organisms,the peptide may have a variety of native forms.

[0380] “Peptide” refers to a polymer in which the monomers are aminoacids and are joined together through amide bonds, alternativelyreferred to as a peptide. Additionally, unnatural amino acids, forexample, β-alanine, phenylglycine and homoarginine are also included.Amino acids that are not nucleic acid-encoded may also be used in thepresent invention. Furthermore, amino acids that have been modified toinclude reactive groups, glycosylation sites, polymers, therapeuticmoieties, biomolecules and the like may also be used in the invention.All of the amino acids used in the present invention may be either theD- or L-isomer thereof. The L-isomer is generally preferred. Inaddition, other peptidomimetics are also useful in the presentinvention. As used herein, “peptide” refers to both glycosylated andunglycosylated peptides. Also included are peptides that areincompletely glycosylated by a system that expresses the peptide. For ageneral review, see, Spatola, A. F., in CHEMISTRY AND BIOCHEMISTRY OFAMINO ACIDS, PEPTIDES AND PROTEINS, B. Weinstein, eds., Marcel Dekker,New York, p. 267 (1983).

[0381] The term “peptide conjugate,” refers to species of the inventionin which a peptide is conjugated with a modified sugar as set forthherein.

[0382] The term “amino acid” refers to naturally occurring and syntheticamino acids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is linked toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that function in amanner similar to a naturally occurring amino acid.

[0383] As used herein, amino acids are represented by the full namethereof, by the three letter code corresponding thereto, or by theone-letter code corresponding thereto, as indicated in the followingTable 1: TABLE 1 Amino acids, and the three letter and one letter codes.Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D GlutamicAcid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineIle I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan TrpW

[0384] The present invention also provides for analogs of proteins orpeptides which comprise a protein as identified above. Analogs maydiffer from naturally occurring proteins or peptides by conservativeamino acid sequence differences or by modifications which do not affectsequence, or by both. For example, conservative amino acid changes maybe made, which although they alter the primary sequence of the proteinor peptide, do not normally alter its function. Conservative amino acidsubstitutions typically include substitutions within the followinggroups:

[0385] glycine, alanine;

[0386] valine, isoleucine, leucine;

[0387] aspartic acid, glutamic acid;

[0388] asparagine, glutamine;

[0389] serine, threonine;

[0390] lysine, arginine;

[0391] phenylalanine, tyrosine.

[0392] Modifications (which do not normally alter primary sequence)include in vivo, or in vitro, chemical derivatization of peptides, e.g.,acetylation, or carboxylation. Also included are modifications ofglycosylation, e.g., those made by modifying the glycosylation patternsof a peptide during its synthesis and processing or in furtherprocessing steps; e.g., by exposing the peptide to enzymes which affectglycosylation, e.g., mammalian glycosylating or deglycosylating enzymes.Also embraced are sequences which have phosphorylated amino acidresidues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

[0393] It will be appreciated, of course, that the peptides mayincorporate amino acid residues which are modified without affectingactivity. For example, the termini may be derivatized to includeblocking groups, i.e. chemical substituents suitable to protect and/orstabilize the N- and C-termini from “undesirable degradation”, a termmeant to encompass any type of enzymatic, chemical or biochemicalbreakdown of the compound at its termini which is likely to affect thefunction of the compound, i.e. sequential degradation of the compound ata terminal end thereof.

[0394] Blocking groups include protecting groups conventionally used inthe art of peptide chemistry which will not adversely affect the in vivoactivities of the peptide. For example, suitable N-terminal blockinggroups can be introduced by alkylation or acylation of the N-terminus.Examples of suitable N-terminal blocking groups include C₁-C₅ branchedor unbranched alkyl groups, acyl groups such as formyl and acetylgroups, as well as substituted forms thereof, such as theacetamidomethyl (Acm), Fmoc or Boc groups. Desamino analogs of aminoacids are also useful N-terminal blocking groups, and can either becoupled to the N-terminus of the peptide or used in place of theN-terminal reside. Suitable C-terminal blocking groups, in which thecarboxyl group of the C-terminus is either incorporated or not, includeesters, ketones or amides. Ester or ketone-forming alkyl groups,particularly lower alkyl groups such as methyl, ethyl and propyl, andamide-forming amino groups such as primary amines (—NH₂), and mono- anddi-alkylamino groups such as methylamino, ethylamino, dimethylamino,diethylamino, methylethylamino and the like are examples of C-terminalblocking groups. Descarboxylated amino acid analogues such as agmatineare also useful C-terminal blocking groups and can be either coupled tothe peptide's C-terminal residue or used in place of it. Further, itwill be appreciated that the free amino and carboxyl groups at thetermini can be removed altogether from the peptide to yield desamino anddescarboxylated forms thereof without affect on peptide activity.

[0395] Other modifications can also be incorporated without adverselyaffecting the activity and these include, but are not limited to,substitution of one or more of the amino acids in the natural L-isomericform with amino acids in the D-isomeric form. Thus, the peptide mayinclude one or more D-amino acid resides, or may comprise amino acidswhich are all in the D-form. Retro-inverso forms of peptides inaccordance with the present invention are also contemplated, forexample, inverted peptides in which all amino acids are substituted withD-amino acid forms.

[0396] Acid addition salts of the present invention are alsocontemplated as functional equivalents. Thus, a peptide in accordancewith the present invention treated with an inorganic acid such ashydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like,or an organic acid such as an acetic, propionic, glycolic, pyruvic,oxalic, malic, malonic, succinic, maleic, fumaric, tataric, citric,benzoic, cinnamic, mandelic, methanesulfonic, ethanesulfonic,p-toluenesulfonic, salicyclic and the like, to provide a water solublesalt of the peptide is suitable for use in the invention.

[0397] Also included are peptides which have been modified usingordinary molecular biological techniques so as to improve theirresistance to proteolytic degradation or to optimize solubilityproperties or to render them more suitable as a therapeutic agent.Analogs of such peptides include those containing residues other thannaturally occurring L-amino acids, e.g., D-amino acids or non-naturallyoccurring synthetic amino acids. The peptides of the invention are notlimited to products of any of the specific exemplary processes listedherein.

[0398] As used herein, the term “MALDI” is an abbreviation for MatrixAssisted Laser Desorption Ionization. During ionization, SA-PEG (sialicacid-poly(ethylene glycol)) can be partially eliminated from theN-glycan structure of the glycoprotein.

[0399] As used herein, the term “glycosyltransferase,” refers to anyenzyme/protein that has the ability to transfer a donor sugar to anacceptor moiety.

[0400] As used herein, the term “modified sugar,” refers to a naturally-or non-naturally-occurring carbohydrate that is enzymatically added ontoan amino acid or a glycosyl residue of a peptide in a process of theinvention. The modified sugar is selected from a number of enzymesubstrates including, but not limited to sugar nucleotides (mono-, di-,and tri-phosphates), activated sugars (e.g., glycosyl halides, glycosylmesylates) and sugars that are neither activated nor nucleotides.

[0401] The “modified sugar” is covalently functionalized with a“modifying group.” Useful modifying groups include, but are not limitedto, water-soluble polymers, therapeutic moieties, diagnostic moieties,biomolecules and the like. The locus of functionalization with themodifying group is selected such that it does not prevent the “modifiedsugar” from being added enzymatically to a peptide.

[0402] The term “water-soluble” refers to moieties that have somedetectable degree of solubility in water. Methods to detect and/orquantify water solubility are well known in the art. Exemplarywater-soluble polymers include peptides, saccharides, poly(ethers),poly(amines), poly(carboxylic acids) and the like. Peptides can havemixed sequences or be composed of a single amino acid, e.g.poly(lysine). Similarly, saccharides can be of mixed sequence orcomposed of a single saccharide subunit, e.g., dextran, amylose,chitosan, and poly(sialic acid). An exemplary poly(ether) ispoly(ethylene glycol). Poly(ethylene imine) is an exemplary polyamine,and poly(aspartic) acid is a representative poly(carboxylic acid).

[0403] “Poly(alkylene oxide)” refers to a genus of compounds having apolyether backbone. Poly(alkylene oxide) species of use in the presentinvention include, for example, straight- and branched-chain species.Moreover, exemplary poly(alkylene oxide) species can terminate in one ormore reactive, activatable, or inert groups. For example, poly(ethyleneglycol) is a poly(alkylene oxide) consisting of repeating ethylene oxidesubunits, which may or may not include additional reactive, activatableor inert moieties at either terminus. Useful poly(alkylene oxide)species include those in which one terminus is “capped” by an inertgroup, e.g., monomethoxy-poly(alkylene oxide). When the molecule is abranched species, it may include multiple reactive, activatable or inertgroups at the termini of the alkylene oxide chains and the reactivegroups may be either the same or different. Derivatives ofstraight-chain poly(alkylene oxide) species that are heterobifunctionalare also known in the art.

[0404] The term, “glycosyl linking group,” as used herein refers to aglycosyl residue to which an agent (e.g., water-soluble polymer,therapeutic moiety, biomolecule) is covalently attached. In the methodsof the invention, the “glycosyl linking group” becomes covalentlyattached to a glycosylated or unglycosylated peptide, thereby linkingthe agent to an amino acid and/or glycosyl residue on the peptide. A“glycosyl linking group” is generally derived from a “modified sugar” bythe enzymatic attachment of the “modified sugar” to an amino acid and/orglycosyl residue of the peptide. More specifically, a “glycosyl linkinggroup,” as used herein, refers to a moiety that covalently joins a“modifying group,” as discussed herein, and an amino acid residue of apeptide. The glycosyl linking group-modifying group adduct has astructure that is a substrate for an enzyme. The enzymes for which theglycosyl linking group-modifying group adduct are substrates aregenerally those capable of transferring a saccharyl moiety onto an aminoacid residue of a peptide, e.g, a glycosyltransferase, amidase,glycosidase, trans-sialidase, etc. The “glycosyl linking group” isinterposed between, and covalently joins a “modifying group” and anamino acid residue of a peptide.

[0405] An “intact glycosyl linking group” refers to a linking group thatis derived from a glycosyl moiety in which the individual saccharidemonomer that links the conjugate is not degraded, e.g., oxidized, e.g.,by sodium metaperiodate. “Intact glycosyl linking groups” of theinvention may be derived from a naturally occurring oligosaccharide byaddition of glycosyl unit(s) or removal of one or more glycosyl unitfrom a parent saccharide structure. An exemplary “intact glycosyllinking group” includes at least one intact, e.g., non-degraded,saccharyl moiety that is covalently attached to an amino acid residue ona peptide. The remainder of the “linking group” can have substantiallyany structure. For example, the modifying group is optionally linkeddirectly to the intact saccharyl moiety. Alternatively, the modifyinggroup is linked to the intact saccharyl moiety via a linker arm. Thelinker arm can have substantially any structure determined to be usefulin the selected embodiment. In an exemplary embodiment, the linker armis one or more intact saccharyl moieties, i.e. “the intact glycosyllinking group” resembles an oligosaccharide. Another exemplary intactglycosyl linking group is one in which a saccharyl moiety attached,directly or indirectly, to the intact saccharyl moiety is degraded andderivatized (e.g., periodate oxidation followed by reductive amination).Still a further linker arm includes the modifying group attached to theintact saccharyl moiety, directly or indirectly, via a cross-linker,such as those described herein or analogues thereof.

[0406] “Degradation,” as used herein refers to the removal of one ormore carbon atoms from a saccharyl moiety.

[0407] The terms “targeting moiety” and “targeting agent”, as usedherein, refer to species that will selectively localize in a particulartissue or region of the body. The localization is mediated by specificrecognition of molecular determinants, molecular size of the targetingagent or conjugate, ionic interactions, hydrophobic interactions and thelike. Other mechanisms of targeting an agent to a particular tissue orregion are known to those of skill in the art.

[0408] As used herein, “therapeutic moiety” means any agent useful fortherapy including, but not limited to, antibiotics, anti-inflammatoryagents, anti-tumor drugs, cytotoxins, and radioactive agents.“Therapeutic moiety” includes prodrugs of bioactive agents, constructsin which more than one therapeutic moiety is linked to a carrier, e.g.,multivalent agents. Therapeutic moiety also includes peptides, andconstructs that include peptides. Exemplary peptides include thosedisclosed in FIG. 28 and Tables 6 and 7, herein. “Therapeutic moiety”thus means any agent useful for therapy including, but not limited to,antibiotics, anti-inflammatory agents, anti-tumor drugs, cytotoxins, andradioactive agents. “Therapeutic moiety” includes prodrugs of bioactiveagents, constructs in which more than one therapeutic moiety is linkedto a carrier, e.g., multivalent agents.

[0409] As used herein, “anti-tumor drug” means any agent useful tocombat cancer including, but not limited to, cytotoxins and agents suchas antimetabolites, alkylating agents, anthracyclines, antibiotics,antimitotic agents, procarbazine, hydroxyurea, asparaginase,corticosteroids, interferons and radioactive agents. Also encompassedwithin the scope of the term “anti-tumor drug,” are conjugates ofpeptides with anti-tumor activity, e.g. TNF-α. Conjugates include, butare not limited to those formed between a therapeutic protein and aglycoprotein of the invention. A representative conjugate is that formedbetween PSGL-1 and TNF-α.

[0410] As used herein, “a cytotoxin or cytotoxic agent” means any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracinedione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Other toxins include, for example, ricin, CC-1065 andanalogues, the duocarmycins. Still other toxins include diphtheriatoxin, and snake venom (e.g., cobra venom).

[0411] As used herein, “a radioactive agent” includes any radioisotopethat is effective in diagnosing or destroying a tumor. Examples include,but are not limited to, indium-111, cobalt-60 and technetium.Additionally, naturally occurring radioactive elements such as uranium,radium, and thorium, which typically represent mixtures ofradioisotopes, are suitable examples of a radioactive agent. The metalions are typically chelated with an organic chelating moiety.

[0412] Many useful chelating groups, crown ethers, cryptands and thelike are known in the art and can be incorporated into the compounds ofthe invention (e.g. EDTA, DTPA, DOTA, NTA, HDTA, etc. and theirphosphonate analogs such as DTPP, EDTP, HDTP, NTP, etc). See, forexample, Pitt et al., “The Design of Chelating Agents for the Treatmentof Iron Overload,” In, INORGANIC CHEMISTRY IN BIOLOGY AND MEDICINE;Martell, Ed.; American Chemical Society, Washington, D.C., 1980, pp.279-312; Lindoy, THE CHEMISTRY OF MACROCYCLIC LIGAND COMPLEXES;Cambridge University Press, Cambridge, 1989; Dugas, BIOORGANICCHEMISTRY; Springer-Verlag, New York, 1989, and references containedtherein.

[0413] Additionally, a manifold of routes allowing the attachment ofchelating agents, crown ethers and cyclodextrins to other molecules isavailable to those of skill in the art. See, for example, Meares et al.,“Properties of In Vivo Chelate-Tagged Proteins and Polypeptides.” In,MODIFICATION OF PROTEINS: FOOD, NUTRITIONAL, AND PHARMACOLOGICALASPECTS;” Feeney, et al., Eds., American Chemical Society, Washington,D.C., 1982, pp. 370-387; Kasina et al., Bioconjugate Chem., 9: 108-117(1998); Song et al., Bioconjugate Chem., 8: 249-255 (1997).

[0414] As used herein, “pharmaceutically acceptable carrier” includesany material, which when combined with the conjugate retains theactivity of the conjugate activity and is non-reactive with thesubject's immune system. Examples include, but are not limited to, anyof the standard pharmaceutical carriers such as a phosphate bufferedsaline solution, water, emulsions such as oil/water emulsion, andvarious types of wetting agents. Other carriers may also include sterilesolutions, tablets including coated tablets and capsules. Typically suchcarriers contain excipients such as starch, milk, sugar, certain typesof clay, gelatin, stearic acid or salts thereof, magnesium or calciumstearate, talc, vegetable fats or oils, gums, glycols, or other knownexcipients. Such carriers may also include flavor and color additives orother ingredients. Compositions comprising such carriers are formulatedby well known conventional methods.

[0415] As used herein, “administering” means oral administration,administration as a suppository, topical contact, intravenous,intraperitoneal, intramuscular, intralesional, intranasal orsubcutaneous administration, intrathecal administration, or theimplantation of a slow-release device e.g., a mini-osmotic pump, to thesubject.

[0416] The term “isolated” refers to a material that is substantially oressentially free from components, which are used to produce thematerial. For peptide conjugates of the invention, the term “isolated”refers to material that is substantially or essentially free fromcomponents, which normally accompany the material in the mixture used toprepare the peptide conjugate. “Isolated” and “pure” are usedinterchangeably. Typically, isolated peptide conjugates of the inventionhave a level of purity preferably expressed as a range. The lower end ofthe range of purity for the peptide conjugates is about 60%, about 70%or about 80% and the upper end of the range of purity is about 70%,about 80%, about 90% or more than about 90%.

[0417] When the peptide conjugates are more than about 90% pure, theirpurities are also preferably expressed as a range. The lower end of therange of purity is about 90%, about 92%, about 94%, about 96% or about98%. The upper end of the range of purity is about 92%, about 94%, about96%, about 98% or about 100% purity.

[0418] Purity is determined by any art-recognized method of analysis(e.g., band intensity on a silver stained gel, polyacrylamide gelelectrophoresis, HPLC, or a similar means).

[0419] “Commercial scale” as used herein means about one or more gram offinal product produced in the method.

[0420] “Essentially each member of the population,” as used herein,describes a characteristic of a population of peptide conjugates of theinvention in which a selected percentage of the modified sugars added toa peptide are added to multiple, identical acceptor sites on thepeptide. “Essentially each member of the population” speaks to the“homogeneity” of the sites on the peptide conjugated to a modified sugarand refers to conjugates of the invention, which are at least about 80%,preferably at least about 90% and more preferably at least about 95%homogenous.

[0421] “Homogeneity,” refers to the structural consistency across apopulation of acceptor moieties to which the modified sugars areconjugated. Thus, in a peptide conjugate of the invention in which eachmodified sugar moiety is conjugated to an acceptor site having the samestructure as the acceptor site to which every other modified sugar isconjugated, the peptide conjugate is said to be about 100% homogeneous.Homogeneity is typically expressed as a range. The lower end of therange of homogeneity for the peptide conjugates is about 60%, about 70%or about 80% and the upper end of the range of purity is about 70%,about 80%, about 90% or more than about 90%.

[0422] When the peptide conjugates are more than or equal to about 90%homogeneous, their homogeneity is also preferably expressed as a range.The lower end of the range of homogeneity is about 90%, about 92%, about94%, about 96% or about 98%. The upper end of the range of purity isabout 92%, about 94%, about 96%, about 98% or about 100% homogeneity.The purity of the peptide conjugates is typically determined by one ormore methods known to those of skill in the art, e.g., liquidchromatography-mass spectrometry (LC-MS), matrix assisted laserdesorption time of flight mass spectrometry (MALDI-TOF), capillaryelectrophoresis, and the like.

[0423] “Substantially uniform glycoform” or a “substantially uniformglycosylation pattern,” when referring to a glycopeptide species, refersto the percentage of acceptor moieties that are glycosylated by theglycosyltransferase of interest (e.g., fucosyltransferase). For example,in the case of a α1,2 fucosyltransferase, a substantially uniformfucosylation pattern exists if substantially all (as defined below) ofthe Galβ1,4-GlcNAc-R and sialylated analogues thereof are fucosylated ina peptide conjugate of the invention. It will be understood by one ofskill in the art, that the starting material may contain glycosylatedacceptor moieties (e.g., fucosylated Galβ1,4-GlcNAc-R moieties). Thus,the calculated percent glycosylation will include acceptor moieties thatare glycosylated by the methods of the invention, as well as thoseacceptor moieties already glycosylated in the starting material.

[0424] The term “substantially” in the above definitions of“substantially uniform” generally means at least about 40%, at leastabout 70%, at least about 80%, or more preferably at least about 90%,and still more preferably at least about 95% of the acceptor moietiesfor a particular glycosyltransferase are glycosylated.

DESCRIPTION OF THE INVENTION

[0425] I. Method to Remodel Glycan Chains

[0426] The present invention includes methods and compositions for thein vitro addition and/or deletion of sugars to or from a glycopeptidemolecule in such a manner as to provide a peptide molecule having aspecific customized or desired glycosylation pattern, preferablyincluding the addition of a modified sugar thereto. A key feature of theinvention therefore is to take a peptide produced by any cell type andgenerate a core glycan structure on the peptide, following which theglycan structure is then remodeled in vitro to generate a peptide havinga glycosylation pattern suitable for therapeutic use in a mammal.

[0427] The importance of the glycosylation pattern of a peptide is wellknown in the art as are the limitations of present in vivo methods forthe production of properly glycosylated peptides, particularly whenthese peptides are produced using recombinant DNA methodology. Moreover,until the present invention, it has not been possible to generateglycopeptides having a desired glycan structure thereon, wherein thepeptide can be produced at industrial scale.

[0428] In the present invention, a peptide produced by a cell isenzymatically treated in vitro by the systematic addition of theappropriate enzymes and substrates therefor, such that sugar moietiesthat should not be present on the peptide are removed, and sugarmoieties, optionally including modified sugars, that should be added tothe peptide are added in a manner to provide a glycopeptide having“desired glycosylation”, as defined elsewhere herein.

[0429] A. Method to Remodel N-Linked Glycans

[0430] In one aspect, the present invention takes advantage of the factthat most peptides of commercial or pharmaceutical interest comprise acommon five sugar structure referred to herein as the trimannosyl core,which is N-linked to asparagine at the sequence Asn-X-Ser/Thr on apeptide chain. The elemental trimannosyl core consists essentially oftwo N-acetylglucosamine (GlcNAc) residues and three mannose (Man)residues attached to a peptide, i.e., it comprises these five sugarresidues and no additional sugars, except that it may optionally includea fucose residue. The first GlcNAc is attached to the amide group of theasparagine and the second GlcNAc is attached to the first via a β1,4linkage. A mannose residue is attached to the second GlcNAc via a β1,4linkage and two mannose residues are attached to this mannose via anα1,3 and an α1,6 linkage respectively. A schematic depiction of atrimannosyl core structure is shown in FIG. 1, left side. While it isthe case that glycan structures on most peptides comprise other sugarsin addition to the trimannosyl core, the trimannosyl core structurerepresents an essential feature of N-linked glycans on mammalianpeptides.

[0431] The present invention includes the generation of a peptide havinga trimannosyl core structure as a fundamental element of the structureof the glycan molecules contained thereon. Given the variety of cellularsystems used to produce peptides, whether the systems are themselvesnaturally occurring or whether they involve recombinant DNA methodology,the present invention provides methods whereby a glycan molecule on apeptide produced in any cell type can be reduced to an elementaltrimannosyl core structure. Once the elemental trimannosyl corestructure has been generated then it is possible using the methodsdescribed herein, to generate in vitro, a desired glycan structure onthe peptide which confers on the peptide one or more properties thatenhances the therapeutic effectiveness of the peptide.

[0432] It should be clear from the discussion herein that the term“trimannosyl core” is used to describe the glycan structure shown inFIG. 1, left side. Glycopeptides having a trimannosyl core structure mayalso have additional sugars added thereto, and for the most part, dohave additional structures added thereto irrespective of whether thesugars give rise to a peptide having a desired glycan structure. Theterm “elemental trimannosyl core structure” is defined elsewhere herein.When the term “elemental” is not included in the description of the“trimannosyl core structure,” then the glycan comprises the trimannosylcore structure with additional sugars attached to the mannose sugars.

[0433] The term “elemental trimannosyl core glycopeptide” is used hereinto refer to a glycopeptide having glycan structures comprised primarilyof an elemental trimannosyl core structure. However, it may alsooptionally contain a fucose residue attached thereto. As discussedherein, elemental trimannosyl core glycopeptides are one optimal, andtherefore preferred, starting material for the glycan remodelingprocesses of the invention.

[0434] Another optimal starting material for the glycan remodelingprocess of the invention is a glycan structure having a trimannosyl corewherein one or two additional GlcNAc residues are added to each of theα1,3 and the α1,6 mannose residues (see for example, the structure onthe second line of FIG. 2, second structure in from the left of thefigure). This structure is referred to herein as “Man3GlcNAc4.” When thestructure is monoantenary, the structure is referred to herein as“Man3GlcNAc3.” Optionally, this structure may also contain a core fucosemolecule. Once the Man3GlcNAc3 or Man3GlcNAc4 structure has beengenerated then it is possible using the methods described herein, togenerate in vitro, a desired glycan structure on the glycopeptide whichconfers on the glycopeptide one or more properties that enhances thetherapeutic effectiveness of the peptide.

[0435] In their native form, the N-linked glycopeptides of theinvention, and particularly the mammalian and human glycopeptides usefulin the present invention, are N-linked glycosylated with a trimannosylcore structure and one or more sugars attached thereto.

[0436] The terms “glycopeptide” and “glycopolypeptide” are usedsynonymously herein to refer to peptide chains having sugar moietiesattached thereto. No distinction is made herein to differentiate smallglycopolypeptides or glycopeptides from large glycopolypeptides orglycopeptides. Thus, hormone molecules having very few amino acids intheir peptide chain (e.g., often as few as three amino acids) and othermuch larger peptides are included in the general terms“glycopolypeptide” and “glycopeptide,” provided they have sugar moietiesattached thereto. However, the use of the term “peptide” does notpreclude that peptide from being a glycopeptide.

[0437] An example of an N-linked glycopeptide having desiredglycosylation is a peptide having an N-linked glycan having atrimannosyl core with at least one GlcNAc residue attached thereto. Thisresidue is added to the trimannosyl core using N-acetylglucosaminyltransferase I (GnT-I). If a second GlcNAc residue is added,N-acetyl glucosaminyltransferase II (GnT-II) is used. Optionally,additional GlcNAc residues may be added with GnT-IV and/or GnT-V, and athird bisecting GlcNAc residue may be attached to the β1,4 mannose ofthe trimannosyl core using N-acetyl glucosaminyltransferase III(GnT-III). Optionally, this structure may be extended by treatment withβ1,4 galactosyltransferase to add a galactose residue to eachnon-bisecting GlcNAc, and even further optionally, using α2,3 orα2,6-sialyltransferase enzymes, sialic acid residues may be added toeach galactose residue. The addition of a bisecting GlcNAc to the glycanis not required for the subsequent addition of galactose and sialic acidresidues; however, with respect to the substrate affinity of the rat andhuman GnT-III enzymes, the presence of one or more of the galactoseresidues on the glycan precludes the addition of the bisecting GlcNAc inthat the galactose-containing glycan is not a substrate for these formsof GnT-II. Thus, in instances where the presence of the bisecting GlcNAcis desired and these forms of GnT-III are used, it is important shouldthe glycan contain added galactose and/or sialic residues, that they areremoved prior to the addition of the bisecting GlcNAc. Other forms ofGnT-III may not require this specific order of substrates for theiractivity. In the more preferred reaction, a mixture of GnT-I, GnT-II andGnT-III is added to the reaction mixture so that the GlcNAc residues canbe added in any order.

[0438] Examples of glycan structures which represent the various aspectsof peptides having “desired glycosylation” are shown in the drawingsprovided herein. The precise procedures for the in vitro generation of apeptide having “desired glycosylation” are described elsewhere herein.However, the invention should in no way be construed to be limitedsolely to any one glycan structure disclosed herein. Rather, theinvention should be construed to include any and all glycan structureswhich can be made using the methodology provided herein.

[0439] In some cases, an elemental trimannosyl core alone may constitutethe desired glycosylation of a peptide. For example, a peptide havingonly a trimannosyl core has been shown to be a useful component of anenzyme employed to treat Gaucher disease (Mistry et al., 1966, Lancet348: 1555-1559; Bijsterbosch et al., 1996, Eur. J. Biochem.237:344-349).

[0440] According to the present invention, the following procedures forthe generation of peptides having desired glycosylation become apparent.

[0441] a) Beginning with a glycopeptide having one or more glycanmolecules which have as a common feature a trimannosyl core structureand at least one or more of a heterogeneous or homogeneous mixture ofone or more sugars added thereto, it is possible to increase theproportion of glycopeptides having an elemental trimannosyl corestructure as the sole glycan structure or which have Man3GlcNAc3 orMan3GlcNAc4 as the sole glycan structure. This is accomplished in vitroby the systematic addition to the glycopeptide of an appropriate numberof enzymes in an appropriate sequence which cleave the heterogeneous orhomogeneous mixture of sugars on the glycan structure until it isreduced to an elemental trimannosyl core or Man3GlcNAc3 or Man3GlcNAc4structure. Specific examples of how this is accomplished will depend ona variety of factors including in large part the type of cell in whichthe peptide is produced and therefore the degree of complexity of theglycan structure(s) present on the peptide initially produced by thecell. Examples of how a complex glycan structure can be reduced to anelemental trimannosyl core or a Man3GlcNAc3 or Man3GlcNAc4 structure arepresented in FIG. 2 or are described in detail elsewhere herein.

[0442] b) It is possible to generate a peptide having an elementaltrimannosyl core structure as the sole glycan structure on the peptideby isolating a naturally occurring cell whose glycosylation machineryproduces such a peptide. DNA encoding a peptide of choice is thentransfected into the cell wherein the DNA is transcribed, translated andglycosylated such that the peptide of choice has an elementaltrimannosyl core structure as the sole glycan structure thereon. Forexample, a cell lacking a functional GnT-I enzyme will produce severaltypes of glycopeptides. In some instances, these will be glycopeptideshaving no additional sugars attached to the trimannosyl core. However,in other instances, the peptides produced may have two additionalmannose residues attached to the trimannosyl core, resulting in a Man5glycan. This is also a desired starting material for the remodelingprocess of the present invention. Specific examples of the generation ofsuch glycan structures are described herein.

[0443] c) Alternatively, it is possible to genetically engineer a cellto confer upon it a specific glycosylation machinery such that a peptidehaving an elemental trimannosyl core or Man3GlcNAc3 or Man3GlcNAc4structure as the sole glycan structure on the peptide is produced. DNAencoding a peptide of choice is then transfected into the cell whereinthe DNA is transcribed, translated and glycosylated such that thepeptide of choice has an increased number of glycans comprising solelyan elemental trimannosyl core structure. For example, certain types ofcells that are genetically engineered to lack GnT-I, may produce aglycan having an elemental trimannosyl core structure, or, depending onthe cell, may produce a glycan having a trimannosyl core plus twoadditional mannose residues attached thereto (Man5). When the cellproduces a Man5 glycan structure, the cell may be further geneticallyengineered to express mannosidase 3 which cleaves off the two additionalmannose residues to generate the trimannosyl core. Alternatively, theMan5 glycan may be incubated in vitro with mannosidase 3 to have thesame effect.

[0444] d) When a peptide is expressed in an insect cell, the glycan onthe peptide comprises a partially complex chain. Insect cells alsoexpress hexosaminidase in the cells which trims the partially complexchain back to a trimannosyl core structure which can then be remodeledas described herein.

[0445] e) It is readily apparent from the discussion in b), c) and d)that it is not necessary that the cells produce only peptides havingelemental trimannosyl core or Man3GlcNAc3 or Man3GlcNAc4 structuresattached thereto. Rather, unless the cells described in b) and c)produce peptides having 100% elemental trimannosyl core structures(i.e., having no additional sugars attached thereto) or 100% ofMan3GlcNAc3 or Man3GlcNAc4 structures, the cells in fact produce aheterogeneous mixture of peptides having, in combination, elementaltrimannosyl core structures, or Man3GlcNAc3 or Man3GlcNAc4 structures,as the sole glycan structure in addition to these structures havingadditional sugars attached thereto. The proportion of peptides having atrimannosyl core or Man3GlcNAc3 or Man3GlcNAc4 structures havingadditional sugars attached thereto, as opposed to those having onestructure, will vary depending on the cell which produces them. Thecomplexity of the glycans (i.e. which and how many sugars are attachedto the trimannosyl core) will also vary depending on the cell whichproduces them.

[0446] f) Once a glycopeptide having an elemental trimannosyl core or atrimannosyl core with one or two GlcNAc residues attached thereto isproduced by following a), b) or c) above, according to the presentinvention, additional sugar molecules are added in vitro to thetrimannosyl core structure to generate a peptide having desiredglycosylation (i.e., a peptide having an in vitro customized glycanstructure).

[0447] g) However, when it is the case that a peptide having anelemental trimannosyl core or Man3GlcNAc4 structure with some but notall of the desired sugars attached thereto is produced, then it is onlynecessary to add any remaining desired sugars without reducing theglycan structure to the elemental trimannosyl core or Man3GlcNAc4structure. Therefore, in some cases, a peptide having a glycan structurehaving a trimannosyl core structure with additional sugars attachedthereto, will be a suitable substrate for remodeling.

[0448] Isolation of an Elemental Trimannosyl Core Glycopeptide

[0449] The elemental trimannosyl core or Man3GlcNAc3 or Man3GlcNAc4glycopeptides of the invention may be isolated and purified, ifnecessary, using techniques well known in the art of peptidepurification. Suitable techniques include chromatographic techniques,isoelectric focusing techniques, ultrafiltration techniques and thelike. Using any such techniques, a composition of the invention can beprepared in which the glycopeptides of the invention are isolated fromother peptides and from other components normally found within cellculture media. The degree of purification can be, for example, 90% withrespect to other peptides or 95%, or even higher, e.g., 98%. See, e.g.,Deutscher et al. (ed., 1990, Guide to Protein Purification, HarcourtBrace Jovanovich, San Diego).

[0450] The heterogeneity of N-linked glycans present in theglycopeptides produced by the prior art methodology generally onlypermits the isolation of a small portion of the target glycopeptideswhich can be modified to produce desired glycopeptides. In the presentmethods, large quantities of elemental trimannosyl core glycopeptidesand other desired glycopeptides, including Man3GlcNAc3 or Man3GlcNAc4glycans, can be produced which can then be further modified to generatelarge quantities of peptides having desired glycosylation.

[0451] Specific enrichment of any particular type of glycan linked to apeptide may be accomplished using lectins which have an affinity for thedesired glycan. Such techniques are well known in the art ofglycobiology.

[0452] A key feature of the invention which is described in more detailbelow, is that once a core glycan structure is generated on any peptide,the glycan structure is then remodeled in vitro to generate a peptidehaving desired glycosylation that has improved therapeutic use in amammal. The mammal may be any type of suitable mammal, and is preferablya human.

[0453] The various scenarios and the precise methods and compositionsfor generating peptides with desired glycosylation will become evidentfrom the disclosure which follows.

[0454] The ultimate objective of the production of peptides fortherapeutic use in mammals is that the peptides should comprise glycanstructures that facilitate rather than negate the therapeutic benefit ofthe peptide. As disclosed throughout the present specification, peptidesproduced in cells may be treated in vitro with a variety of enzymeswhich catalyze the cleavage of sugars that should not be present on theglycan and the addition of sugars which should be present on the glycansuch that a peptide having desired glycosylation and thus suitable fortherapeutic use in mammals is generated. The generation of differentglycoforms of peptides in cells is described above. A variety ofmechanisms for the generation of peptides having desired glycosylationis now described, where the starting material i.e., the peptide producedby a cell may differ from one cell type to another. As will becomeapparent from the present disclosure, it is not necessary that thestarting material be uniform with respect to its glycan composition.However, it is preferable that the starting material be enriched forcertain glycoforms in order that large quantities of end product, i.e.,correctly glycosylated peptides are produced.

[0455] In a preferred embodiment according to the present invention, thedegradation and synthesis events that result in a peptide having desiredglycosylation involve at some point, the generation of an elementaltrimannosyl core structure or a Man3GlcNAc3 or Man3GlcNAc4 structure onthe peptide.

[0456] The present invention also provides means of adding one or moreselected glycosyl residues to a peptide, after which a modified sugar isconjugated to at least one of the selected glycosyl residues of thepeptide. The present embodiment is useful, for example, when it isdesired to conjugate the modified sugar to a selected glycosyl residuethat is either not present on a peptide or is not present in a desiredamount. Thus, prior to coupling a modified sugar to a peptide, theselected glycosyl residue is conjugated to the peptide by enzymatic orchemical coupling. In another embodiment, the glycosylation pattern of apeptide is altered prior to the conjugation of the modified sugar by theremoval of a carbohydrate residue from the peptide. See for example WO98/31826.

[0457] Addition or removal of any carbohydrate moieties present on thepeptide is accomplished either chemically or enzymatically. Chemicaldeglycosylation is preferably brought about by exposure of the peptidevariant to the compound trifluoromethanesulfonic acid, or an equivalentcompound. This treatment results in the cleavage of most or all sugarsexcept the linking sugar (N-acetylglucosamine or N-acetylgalactosamine),while leaving the peptide intact. Chemical deglycosylation is describedby Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259: 52 and by Edgeet al., 1981, Anal. Biochem. 118: 131. Enzymatic cleavage ofcarbohydrate moieties on peptide variants can be achieved by the use ofa variety of endo- and exo-glycosidases as described by Thotakura etal., 1987, Meth. Enzymol. 138: 350.

[0458] Chemical addition of glycosyl moieties is carried out by anyart-recognized method. Enzymatic addition of sugar moieties ispreferably achieved using a modification of the methods set forthherein, substituting native glycosyl units for the modified sugars usedin the invention. Other methods of adding sugar moieties are disclosedin U.S. Pat. Nos. 5,876,980, 6,030,815, 5,728,554, and 5,922,577.

[0459] Exemplary attachment points for selected glycosyl residueinclude, but are not limited to: (a) sites for N- and O-glycosylation;(b) terminal glycosyl moieties that are acceptors for aglycosyltransferase; (c) arginine, asparagine and histidine; (d) freecarboxyl groups; (e) free sulfhydryl groups such as those of cysteine;(f) free hydroxyl groups such as those of serine, threonine, orhydroxyproline; (g) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan; or (h) the amide group of glutamine. Exemplarymethods of use in the present invention are described in WO 87/05330published Sep. 11, 1987, and in Aplin and Wriston, CRC Crit. Rev.Biochem., pp. 259-306 (1981).

[0460] Dealing specifically with the examples shown in several of thefigures provided herein, a description of the sequence of in vitroenzymatic reactions for the production of desired glycan structures onpeptides is now presented. The precise reaction conditions for each ofthe enzymatic conversions disclosed below are well known to thoseskilled in the art of glycobiology and are therefore not repeated here.For a review of the reaction conditions for these types of reactions,see Sadler et al., 1982, Methods in Enzymology 83:458-514 and referencescited therein.

[0461] In FIG. 1 there is shown the structure of an elementaltrimannosyl core glycan on the left side. It is possible to convert thisstructure to a complete glycan structure having a bisecting GlcNAc byincubating the elemental trimannosyl core structure in the presence ofGnT-I, followed by GnT-II, and further followed by GnT-III, and a sugardonor comprising UDP-GlcNAc, wherein GlcNAc is sequentially added to theelemental trimannosyl core structure to generate a trimannosyl corehaving a bisecting GlcNAc. In some instances, for example whenremodeling Fc glycans as described herein, the order of addition ofGnT-I, GnT-II and GnT-III may be contrary to that reported in theliterature. The bisecting GlcNAc structure may be produced by adding amixture of GnT-I, GnT-II and GnT-III and UDP-GlcNAc to the reactionmixture

[0462] In FIG. 3 there is shown the conversion of a bisecting GlcNAccontaining trimannosyl core glycan to a complex glycan structurecomprising galactose and N-acetyl neuraminic acid. The bisecting GlcNAccontaining trimannosyl core glycan is first incubated withgalactosyltransferase and UDP-Gal as a donor molecule, wherein twogalactose residues are added to the peripheral GlcNAc residues on themolecule. The enzyme NeuAc-transferase is then used to add two NeuAcresidues one to each of the galactose residues.

[0463] In FIG. 4 there is shown the conversion of a high mannose glycanstructure to an elemental trimannosyl core glycan. The high mannoseglycan (Man9) is incubated sequentially in the presence of themannosidase 1 to generate a Man5 structure and then in the presence ofmannosidase 3, wherein all but three mannose residues are removed fromthe glycan. Alternatively, incubation of the Man9 structure may betrimmed back to the trimannosyl core structure solely by incubation inthe presence of mannosidase 3. According to the schemes presented inFIGS. 1 and 3 above, conversion of this elemental trimannosyl coreglycan to a complex glycan molecule is then possible.

[0464] In FIG. 5 there is shown a typical complex N-linked glycanstructure produced in plant cells. It is important to note that whenplant cells are deficient in GnT-I enzymatic activity, xylose and fucosecannot be added to the glycan. Thus, the use of GnT-I knock-out cellsprovides a particular advantage in the present invention in that thesecells produce peptides having an elemental trimannosyl core onto whichadditional sugars can be added without performing any “trimming back”reactions. Similarly, in instances where the structure produced in aplant cell may be of the Man5 variety of glycan, if GnT-I is absent inthese cells, xylose and fucose cannot be added to the structure. In thiscase, the Man5 structure may be trimmed back to an elemental trimannosylcore (Man3) using mannosidase 3. According to the methods providedherein, it is now possible to add desired sugar moieties to thetrimannosyl core to generate a desired glycan structure.

[0465] In FIG. 6 there is shown a typical complex N-linked glycanstructure produced in insect cells. As is evident, additional sugars,such as, for example, fucose may also be present. Further although notshown here, insect cells may produce high mannose glycans having as manyas nine mannose residues and may have additional sugars attachedthereto. It is also the case in insect cells that GnT-I knock out cellsprevent the addition of fucose residues to the glycan. Thus, productionof a peptide in insect cells may preferably be accomplished in a GnT-Iknock out cell. The glycan thus produced may then be trimmed back invitro if necessary using any of the methods and schemes describedherein, and additional sugars may be added in vitro thereto also usingthe methods and schemes provided herein.

[0466] In FIG. 2 there is shown glycan structures in various stages ofcompletion. Specifically, the in vitro enzymatic generation of anelemental trimannosyl core structure from a complex carbohydrate glycanstructure which does not contain a bisecting GlcNAc residue is shown.Also shown is the generation of a glycan structure therefrom whichcontains a bisecting GlcNAc. Several intermediate glycan structureswhich can be produced are shown. These structures can be produced bycells, or can be produced in the in vitro trimming back reactionsdescribed herein. Sugar moieties may be added in vitro to the elementaltrimannosyl core structure, or to any suitable intermediate structure inorder that a desired glycan is produced.

[0467] In FIG. 7 there is shown a series of possible in vitro reactionswhich can be performed to trim back and add onto glycans beginning witha high mannose structure. For example, a Man9 glyean may be trimmedusing mannosidase 1 to generate a Man5 glycan, or it may be trimmed to atrimannosyl core using mannosidase 3 or one or more microbialmannosidases. GnT-I and or GnT-II may then be used to transferadditional GlcNAc residues onto the glycan. Further, there is shown thesituation which would not occur when the glycan molecule is produced ina cell that does not have GnT-I (see shaded box). For example, fucoseand xylose may be added to a glycan only when GnT-I is active andfacilitates the transfer of a GlcNAc to the molecule.

[0468]FIG. 8 depicts well known strategies for the synthesis ofbiantennary, triantennary and even tetraantennary glycan structuresbeginning with the trimannosyl core structure. According to the methodsof the invention, it is possible to synthesize each of these structuresin vitro using the appropriate enzymes and reaction conditions wellknown in the art of glycobiology.

[0469]FIG. 9 depicts two methods for synthesis of a monoantennary glycanstructure beginning from a high mannose (6 to 9 mannose moieties) glycanstructures. A terminal sialic acid-PEG moiety may be added in place ofthe sialic acid moiety in accordance with glycoPEGylation methodologydescribed herein. In the first method, endo-H is used to cleave theglycan structure on the peptide back to the first GlcNAc residue.Galactose is then added using galactosyltransferase and sialylated-PEGis added as described elsewhere herein. In the second method,mannosidase I is used to cleave mannose residues from the glycanstructure in the peptide. A galactose residue is added to one arm of theremaining mannose residues which were cleaved off the glycan using JackBean α-mannosidase. Sialylated-PEG is then added to this structure asdirected.

[0470]FIG. 10 depicts two additional methods for synthesis of amonoantennary glycan structures beginning from high mannose (6 to 9mannose moieties) glycan structure. As in FIG. 9, a terminal sialicacid-PEG moiety may be added in place of the sialic acid moiety inaccordance with the glycoPEGylation methodology described herein. In thesituation described here, some of the mannose residues from the arm towhich sialylated-PEG is not added, are removed.

[0471] In FIG. 11 there is shown a scheme for the synthesis of yet morecomplex carbohydrate structures beginning with a trimannosyl corestructure. For example, a scheme for the in vitro production of Lewis xand Lewis a antigen structures, which may or may not be sialylated isshown. Such structures when present on a peptide may confer on thepeptide immunological advantages for upregulating or downregulating theimmune response. In addition, such structures are useful for targetingthe peptide to specific cells, in that these types of structures areinvolved in binding to cell adhesion peptides and the like.

[0472]FIG. 12 is an exemplary scheme for preparing an array of O-linkedpeptides originating with serine or threonine.

[0473]FIG. 13 is a series of diagrams depicting the four types ofO-linked glycan structure termed cores 1 through 4. The core structureis outlined in dotted lines. Sugars which may also be included in thisstructure include sialic acid residues added to the galactose residues,and fucose residues added to the GlcNAc residues.

[0474] Thus, in preferred embodiments, the present invention provides amethod of making an N-linked glycosylated glycopeptide by providing anisolated and purified glycopeptide to which is attached an elementaltrimannosyl core or a Man3GlcNAc4 structure, contacting the glycopeptidewith a glycosyltransferase enzyme and a donor molecule having a glycosylmoiety under conditions suitable to transfer the glycosyl moiety to theglycopeptide. Customization of a trimannosyl core glycopeptide orMan3GlcNAc4 glycopeptide to produce a peptide having a desiredglycosylation pattern is then accomplished by the sequential addition ofthe desired sugar moieties, using techniques well known in the art.

[0475] Determination of Glycan Primary Structure

[0476] When an N-linked glycopeptide is produced by a cell, as notedelsewhere herein, it may comprise a heterogeneous mixture of glycanstructures which must be reduced to a common, generally elementaltrimannosyl core or Man3GlcNAc4 structure, prior to adding other sugarmoieties thereto. In order to determine exactly which sugars should beremoved from any particular glycan structure, it is sometimes necessarythat the primary glycan structure be identified. Techniques for thedetermination of glycan primary structure are well know in the art andare described in detail, for example, in Montreuil, “Structure andBiosynthesis of Glycopeptides” In Polysaccharides in MedicinalApplications, pp. 273-327, 1996, Eds. Severian Damitriu, Marcel Dekker,NY. It is therefore a simple matter for one skilled in the art ofglycobiology to isolate a population of peptides produced by a cell anddetermine the structure(s) of the glycans attached thereto. For example,efficient methods are available for (i) the splitting of glycosidicbonds either by chemical cleavage such as hydrolysis, acetolysis,hydrazinolysis, or by nitrous deamination; (ii) complete methylationfollowed by hydrolysis or methanolysis and by gas-liquid chromatographyand mass spectroscopy of the partially methylated monosaccharides; and(iii) the definition of anomeric linkages between monosaccharides usingexoglycosidases, which also provide insight into the primary glycanstructure by sequential degradation. In particular, the techniques ofmass spectroscopy and nuclear magnetic resonance (NMR) spectrometry,especially high field NMR have been successfully used to determineglycan primary structure.

[0477] Kits and equipment for carbohydrate analysis are alsocommercially available. Fluorophore Assisted CarbohydrateElectrophoresis (FACE®) is available from Glyko, Inc. (Novato, Calif.).In FACE analysis, glycoconjugates are released from the peptide witheither Endo H or N-glycanase (PNGase F) for N-linked glycans, orhydrazine for Ser/Thr linked glycans. The glycan is then labeled at thereducing end with a fluorophore in a non-structure discriminatingmanner. The fluorophore labeled glycans are then separated inpolyacrylamide gels based on the charge/mass ratio of the saccharide aswell as the hydrodynamic volume. Images are taken of the gel under UVlight and the composition of the glycans are determined by the migrationdistance as compared with the standards. Oligosaccharides can besequenced in this manner by analyzing migration shifts due to thesequential removal of saccharides by exoglycosidase digestion.

[0478] Exemplary Embodiment

[0479] The remodeling of N-linked glycosylation is best illustrated withreference to Formula 1:

[0480] where X³, X⁴, X⁵, X⁶, X⁷ and X¹⁷ are (independently selected)monosaccharide or oligosaccharide residues; and

[0481] a, b, c, d, e and x are (independently selected) 0, 1 or 2, withthe proviso that at least one member selected from a, b, c, d, e and xare 1 or 2.

[0482] Formula 1 describes glycan structure comprising the tri-mannosylcore, which is preferably covalently linked to an asparagine residue ona peptide backbone. Preferred expression systems will express andsecrete exogenous peptides with N-linked glycans comprising thetri-mannosyl core. Using the remodeling method of the invention, theglycan structures on these peptides can be conveniently remodeled to anyglycan structure desired. Exemplary reaction conditions are foundthroughout the examples and in the literature.

[0483] In preferred embodiments, the glycan structures are remodeled sothat the structure described in Formula 1 has specific determinates. Thestructure of the glycan can be chosen to enhance the biological activityof the peptide, give the peptide a new biological activity, remove thebiological activity of peptide, or better approximate the glycosylationpattern of the native peptide, among others.

[0484] In the first preferred embodiment, the peptide N-linked glycansare remodeled to better approximate the glycosylation pattern of nativehuman proteins. In this embodiment, the glycan structure described inFormula 1 is remodeled to have the following moieties:

[0485] X³ and X⁵=|-GlcNAc-Gal-SA;

[0486] a and c=1;

[0487] d=0 or 1;

[0488] b, e and x=0.

[0489] This embodiment is particularly advantageous for human peptidesexpressed in heterologous cellular expression systems. By remodeling theN-linked glycan structures to this configuration, the peptide can bemade less immunogenic in a human patient, and/or more stable, amongothers.

[0490] In the second preferred embodiment, the peptide N-linked glycansare remodeled to have a bisecting GlcNAc residue on the tri-mannosylcore. In this embodiment, the glycan structure described in Formula 1 isremodeled to have the following moieties:

[0491] X³ and X⁵ are |-GlcNAc-Gal-SA;

[0492] a and c=1;

[0493] X⁴ is GlcNAc;

[0494] b=1;

[0495] d=0 or 1;

[0496] e and x=0.

[0497] This embodiment is particularly advantageous for recombinantantibody molecules expressed in heterologous cellular systems. When theantibody molecule includes a Fc-mediated cellular cytotoxicity, it isknown that the presence of bisected oligosaccharides linked the Fcdomain dramatically increased antibody-dependent cellular cytotoxicity.

[0498] In a third preferred embodiment, the peptide N-linked glycans areremodeled to have a sialylated Lewis X moiety. In this embodiment, theglycan structure described in Formula 1 is remodeled to have thefollowing moieties:

[0499] a, c, d=1;

[0500] b, e and x=0;

[0501] X⁶=fucose.

[0502] This embodiment is particularly advantageous when the peptidewhich is being remodeling is intended to be targeted to selectinmolecules and cells exhibiting the same.

[0503] In a fourth preferred embodiment, the peptide N-linked glycansare remodeled to have a conjugated moiety. The conjugated moiety may bea PEG molecule, another peptide, a small molecule such as a drug, amongothers. In this embodiment, the glycan structure described in Formula 1is remodeled to have the following moieties:

[0504] X³ and X⁵ are |-GlcNAc-Gal-SA-R;

[0505] a and c=1 or 2;

[0506] d=0 or 1;

[0507] b, d, e and x=0;

[0508] where R=conjugate group.

[0509] The conjugated moiety may be a PEG molecule, another peptide, asmall molecule such as a drug, among others. This embodiment thereforeis useful for conjugating the peptide to PEG molecules that will slowthe clearance of the peptide from the patient's bloodstream, to peptidesthat will target both peptides to a specific tissue or cell, or toanother peptide of complementary therapeutic use.

[0510] It will be clear to one of skill in the art that the invention isnot limited to the preferred glycan molecules described above. Thepreferred embodiments are only a few of the many useful glycan moleculesthat can be made by the remodeling method of the invention. Thoseskilled in the art will know how to design other useful glycans.

[0511] In the first exemplary embodiments, the peptide is expressed in aCHO (Chinese hamster ovarian cell line) according to methods well knownin the art. When a peptide with N-linked glycan consensus sites isexpressed and secreted from CHO cells, the N-linked glycans will havethe structures depicted in top row of FIG. 2, but also comprising a corefucose. While all of these structures may be present, by far the mostcommon structures are the two at the right side. In the terms of Formula1,

[0512] X³ and X⁵ are |-GlcNAc-Gal-(SA);

[0513] a and c=1;

[0514] b, e and x=0, and

[0515] d=0 or 1.

[0516] Therefore, in one exemplary embodiment, the N-linked glycans ofpeptides expressed in CHO cells are remodeled to the preferred humanizedglycan by contacting the peptides with a glycosyltransferase that isspecific for a galactose acceptor molecule and a sialic acid donormolecule. This process is illustrated in FIG. 2 and Example 17. Inanother exemplary embodiment, the N-linked glycans of a peptideexpressed and secreted from CHO cells are remodeled to be the preferredPEGylated structures. The peptide is first contacted with a glycosidasespecific for sialic acid to remove the terminal SA moiety, and thencontacted with a glycosyltransferase specific for a galactose acceptormoiety and an sialic acid acceptor moiety, in the presence of PEG-sialicacid-nucleotide donor molecules. Optionally, the peptide may then becontacted with a glycosyltransferase specific for a galactose acceptormoiety and an sialic acid acceptor moiety, in the presence of sialicacid-nucleotide donor molecules to ensure complete the SA capping of allof the glycan molecules.

[0517] In other exemplary embodiments, the peptide is expressed ininsect cells, such as the sf9 cell line, according to methods well knownin the art. When a peptide with N-linked glycan consensus sites isexpressed and secreted from sf9 cells, the N-linked glycans will oftenhave the structures depicted in top row of FIG. 6. In the terms ofFormula 1:

[0518] X³ and X⁵ are |-GlcNAc;

[0519] a and c=0 or 1;

[0520] b=0;

[0521] X⁶ is fucose,

[0522] d=0, 1 or 2; and

[0523] e and x=0.

[0524] The trimannose core is present in the vast majority of theN-linked glycans made by insect cells, and sometimes an antennary GlcNAcand/or fucose residue(s) are also present. Note that the glycan may haveno core fucose, it may have a single core fucose having either linkage,or it may have a single core fucose with a perponderance of a singlelinkage. In one exemplary embodiment, the N-linked glycans of a peptideexpressed and secreted from insect cells is remodeled to the preferredhumanized glycan by first contacting the glycans with a glycosidasespecific to fucose molecules, then contacting the glycans with aglycosyltransferases specific to the mannose acceptor molecule on eachantennary of the trimannose core, a GlcNAc donor molecule in thepresence of nucleotide-GlcNAc molecules; then contacting the glycanswith a glycosyltransferase specific to a GlcNAc acceptor molecule, a Galdonor molecule in the presence of nucleotide-Gal molecules; and thencontacting the glycans with a glycosyltransferase specific to agalactose acceptor molecule, a sialic acid donor molecule in thepresence of nucleotide-SA molecules. One of skill in the art willappreciate that the fucose molecules, if any, can be removed at any timeduring the procedure, and if the core fucose is of the same alpha 1,6linkage as found in human glycans, it may be left intact. In anotherexemplary embodiment, the humanized glycan of the previous example isremodeled further to the sialylated Lewis X glycan by contacting theglycan further with a glycosyltransferase specific to a GlcNAc acceptormolecule, a fucose donor molecule in the presence of nucleotide-fucosemolecules. This process is illustrated in FIG. 11 and Example 39.

[0525] In yet other exemplary embodiments, the peptide is expressed inyeast, such as Saccharomyces cerevisiae, according to methods well knownin the art. When a peptide with N-linked glycan consensus sites isexpressed and secreted from S. cerevisiae cells, the N-linked glycanswill have the structures depicted at the left in FIG. 4. The N-linkedglycans will always have the trimannosyl core, which will often beelaborated with mannose or related polysaccharides of up to 1000residues. In the terms of Formula 1:

[0526] X³ and X⁵=1-Man-Man-(Man)₀₋₁₀₀₀;

[0527] a and c=1 or 2;

[0528] b, d, e and x=0.

[0529] In one exemplary embodiment, the N-linked glycans of a peptideexpressed and secreted from yeast cells are remodeled to the elementaltrimannose core by first contacting the glycans with a glycosidasespecific to α2 mannose molecules, then contacting the glycans with aglycosidase specific to α6 mannose molecules. This process isillustrated in FIG. 4 and Example 38.

[0530] In another exemplary embodiment, the N-linked glycans are furtherremodeled to make a glycan suitable for an recombinant antibody withFc-mediated cellular toxicity function by contacting the elementaltrimannose core glycans with a glycosyltransferase specific to themannose acceptor molecule on each antennary of the trimannose core and aGlcNAc donor molecule in the presence of nucleotide-GlcNAc molecules.Then, the glycans are contacted with a glycosyltransferase specific tothe acceptor mannose molecule in the middle of the trimannose core, aGlcNAc donor molecule in the presence of nucleotide-GlcNAc molecules andfurther contacting the glycans with a glycosyltransferase specific to aGlcNAc acceptor molecule, a Gal donor molecule in the presence ofnucleotide-Gal molecules; and then optionally contacting the glycanswith a glycosyltransferase specific to a galactose acceptor molecule andfurther optionally a sialic acid donor molecule in the presence ofnucleotide-SA molecules. This process is illustrated in FIGS. 1, 2 and3.

[0531] In another exemplary embodiment, the peptide is expressed inbacterial cells, in particular E. coli cells, according to methods wellknown in the art. When a peptide with N-linked glycans consensus sitesis expressed in E. coli cells, the N-linked consensus sites will not beglycosylated. In an exemplary embodiment, a humanized glycan molecule isbuilt out from the peptide backbone by contacting the peptides with aglycosyltransferase specific for a N-linked consensus site and a GlcNAcdonor molecule in the presence of nucleotide-GlcNAc; and furthersequentially contacting the growing glycans with glycosyltransferasesspecific for the acceptor and donor moieties in the present of therequired donor moiety until the desired glycan structure is completed.When a peptide with N-linked glycans is expressed in a eukaryotic cellsbut without the proper leader sequences that direct the nascent peptideto the golgi apparatus, the mature peptide is likely not to beglycosylated. In this case as well the peptide may be given N-linkedglycosylation by building out from the peptide N-linked consensus siteas aforementioned. When a protein is chemically modified with a sugarmoiety, it can be built out as aforementioned.

[0532] These examples are meant to illustrate the invention, and not tolimit it. One of skill in the art will appreciate that the steps takenin each example may in some circumstances be able to be performed in adifferent order to get the same result. One of skill in the art willalso understand that a different set of steps may also produce the sameresulting glycan. The preferred remodeled glycan is by no means specificto the expression system that the peptide is expressed in. The remodeledglycans are only illustrative and one of skill in the art will know howto take the principles from these examples and apply them to peptidesproduced in different expression systems to make glycans notspecifically described herein.

[0533] B. Method to Remodel O-Linked Glycans

[0534] O-glycosylation is characterized by the attachment of a varietyof monosaccharides in an O-glycosidic linkage to hydroxy amino acids.O-glycosylation is a widespread post-translational modification in theanimal and plant kingdoms. The structural complexity of glycans O-linkedto proteins vastly exceeds that of N-linked glycans. Serine or threonineresidues of a newly translated peptide become modified by virtue of apeptidyl GalNAc transferase in the cis to trans compartments of theGolgi. The site of O-glycosylation is determined not only by thesequence specificity of the glycosyltransferase, but also epigeneticregulation mediated by competition between different substrate sites andcompetition with other glycosyltransferases responsible for forming theglycan.

[0535] The O-linked glycan has been arbitrarily defined as having threeregions: the core, the backbone region and the peripheral region. The“core” region of an O-linked glycan is the inner most two or threesugars of the glycan chain proximal to the peptide. The backbone regionmainly contributes to the length of the glycan chain formed by uniformelongation. The peripheral region exhibits a high degree of structuralcomplexity. The structural complexity of the O-linked glycans beginswith the core structure. In most cases, the first sugar residue added atthe O-linked glycan consensus site is GalNAc; however the sugar may alsobe GlcNAc, glucose, mannose, galactose or fucose, among others. FIG. 12is a diagram of some of the known O-linked glycan core structures andthe enzymes responsible for their in vivo synthesis.

[0536] In mammalian cells, at least eight different O-linked corestructures are found, all based on a core-α-GalNAc residue. The fourcore structures depicted in FIG. 13 are the most common. Core 1 and core2 are the most abundant structures in mammalian cells, and core 3 andcore 4 are found in more restricted, organ-characteristic expressionsystems. O-linked glycans are reviewed in Montreuil, Structure andSynthesis of Glycopeptides, In Polysaccharides in MedicinalApplications, pp. 273-327, 1996, Eds. Severian Damitriu, Marcel Dekker,NY, and in Schachter and Brockhausen, The Biosynthesis of BranchedO-Linked Glycans, 1989, Society for Experimental Biology, pp. 1-26(Great Britain).

[0537] It will be apparent from the present disclosure that the glycanstructure of O-glycosylated peptides can be remodeled using similartechniques to those described for N-linked glycans. O-glycans differfrom N-glycans in that they are linked to a serine or threonine residuerather than an asparagine residue. As described herein with respect toN-glycan remodeling, hydrolytic enzymes can be used to cleave unwantedsugar moieties in an O-linked glycan and additional desired sugars canthen be added thereto, to build a customized O-glycan structure on thepeptide (See FIGS. 12 and 13).

[0538] The initial step in O-glycosylation in mammalian cells is theattachment of N-acetylgalactosamine (GalNAc) using any of a family of atleast eleven known α-N-acetylgalactosaminyltransferases, each of whichhas a restricted acceptor peptide specificity. Generally, the acceptorpeptide recognized by each enzyme constitutes a sequence of at least tenamino acids. Peptides that contain the amino acid sequence recognized byone particular GalNAc-transferase become O-glycosylated at the acceptorsite if they are expressed in a cell expressing the enzyme and if theyare appropriately localized to the Golgi apparatus where UDP-GalNAc isalso present.

[0539] However, in the case of recombinant proteins, the initialattachment of the GalNAc may not take place. Theα-N-acetylgalactosaminyltransferase enzyme native to the expressing cellmay have a consensus sequence specificity which differs from that of therecombinant peptide being expressed.

[0540] The desired recombinant peptide may be expressed in a bacterialcell, such as E. coli, that does not synthesize glycan chains. In thesecases, it is advantageous to add the initial GalNAc moiety in vitro. TheGalNAc moiety can be introduced in vitro onto the peptide once therecombinant peptide has been recovered in a soluble form, by contactingthe peptide with the appropriate GalNAc transferase in the presence ofUDP-GalNAc.

[0541] In one embodiment, an additional sequence of amino acids thatconstitute an effective acceptor for transfer of an O-linked sugar maybe present. Such an amino acid sequence is encoded by a DNA sequencefused in frame to the coding sequence of the peptide, or alternatively,may be introduced by chemical means. The peptide may be otherwiselacking glycan chains. Alternately, the peptide may have N- and/orO-linked glycan chains but require an additional glycosylation site, forexample, when an additional glycan substituent is desired.

[0542] In an exemplary embodiment, the amino acid sequence PTTTK-COOH,which is the natural GalNAc acceptor sequence in the human mucin MUC-1,is added as a fusion tag. The fusion protein is then expressed in E.coli and purified. The peptide is then contacted with recombinant humanGalNAc-transferases T3 or T6 in the presence of UDP-GalNAc to transfer aGalNAc residue onto the peptide in vitro.

[0543] This glycan chain on the peptide may then be further elongatedusing the methods described in reference to the N-linked or O-linkedglycans herein. Alternatively, the GalNAc transferase reaction can becarried out in the presence of UDP-GalNAc to which PEG is covalentlysubstituted in the O-3, 4, or 6 positions or the N-2 position.Glycoconjugation is described in detail elswhere herein. Anyantigenicity introduced into the peptide by the new peptide sequence canbe conveniently masked by PEGylation of the associated glycan. Theacceptor site fusion technique can be used to introduce not only a PEGmoiety, but to introduce other glycan and non-glycan moieties,including, but not limited to, toxins, anti-infectives, cytotoxicagents, chelators for radionucleotides, and glycans with otherfunctionalities, such as tissue targeting.

[0544] Exemplary Embodiments

[0545] The remodeling of O-linked glycosylation is best illustrated withreference to Formula 2:

[0546] Formula 2 describes a glycan structure comprising a GalNAc whichis covalently linked preferably to a serine or threonine residue on apeptide backbone. While this structure is used to illustrate the mostcommon forms of O-linked glycans, it should not be construed to limitthe invention solely to these O-linked glycans. Other forms of O-linkedglycans are illustrated in FIG. 12. Preferred expression systems usefulin the present invention express and secrete exogenous peptides havingO-linked glycans comprising the GalNAc residue. Using the remodelingmethods of the invention, the glycan structures on these peptides can beconveniently remodeled to generate any desired glycan structure. One ofskill in the art will appreciate that O-linked glycans can be remodeledusing the same principles, enzymes and reaction conditions as thoseavailable in the art once armed with the present disclosure. Exemplaryreaction conditions are found throughout the Examples.

[0547] In preferred embodiments, the glycan structures are remodeled sothat the structure described in Formula 2 has specific moieties. Thestructure of the glycan may be chosen to enhance the biological activityof the peptide, confer upon the peptide a new biological activity,remove or alter a biological activity of peptide, or better approximatethe glycosylation pattern of the native peptide, among others.

[0548] In the first preferred embodiment, the peptide O-linked glycansare remodeled to better approximate the glycosylation pattern of nativehuman proteins. In this embodiment, the glycan structure described inFormula 2 is remodeled to have the following moieties:

[0549] X² is |-SA; or |-SA-SA;

[0550] f and n=0 or 1;

[0551] X¹⁰ is SA;

[0552] m=0.

[0553] This embodiment is particularly advantageous for human peptidesexpressed in heterologous cellular expression systems. By remodeling theO-linked glycan structures to have this configuration, the peptide canbe rendered less immunogenic in a human patient and/or more stable.

[0554] In the another preferred embodiment, the peptide O-linked glycansare remodeled to display a sialylated Lewis X antigen. In thisembodiment, the glycan structure described in Formula 2 is remodeled tohave the following moieties:

[0555] X² is |-SA;

[0556] X¹⁰ is Fuc or |-GlcNAc(Fuc)-Gal-SA;

[0557] f and n=1;

[0558] m=0.

[0559] This embodiment is particularly advantageous when the peptidewhich is being remodeled is most effective when targeted to a selectinmolecule and cells exhibiting the same.

[0560] In a yet another preferred embodiment, the peptide O-linkedglycans are remodeled to contain a conjugated moiety. The conjugatedmoiety may be a PEG molecule, another peptide, a small molecule such asa drug, among others. In this embodiment, the glycan structure describedin Formula 2 is remodeled to have the following moieties:

[0561] X² is |-SA-R;

[0562] f=1;

[0563] n and m=0;

[0564] where R is the conjugate group.

[0565] This embodiment is useful for conjugating the peptide to PEGmolecules that will slow the clearance of the peptide from the patient'sbloodstream, to peptides that will target both peptides to a specifictissue or cell or to another peptide of complementary therapeutic use.

[0566] It will be clear to one of skill in the art that the invention isnot limited to the preferred glycan molecules described above. Thepreferred embodiments are only a few of the many useful glycan moleculesthat can be made using the remodeling methods of the invention. Thoseskilled in the art will know how to design other useful glycans oncearmed with the present invention.

[0567] In the first exemplary embodiment, the peptide is expressed in aCHO (Chinese hamster cell line) according to methods well known in theart. When a peptide with O-linked glycan consensus sites is expressedand secreted from CHO cells, the majority of the O-linked glycans willoften have the structure, in the terms of Formula 2,

[0568] X²|-SA;

[0569] f=1;

[0570] m and n=0.

[0571] Therefore, most of the glycans in CHO cells do not requireremodeling in order to be acceptable for use in a human patient. In anexemplary embodiment, the O-linked glycans of a peptide expressed andsecreted from a CHO cell are remodeled to contain a sialylated Lewis Xstructure by contacting the glycans with a glycosyltransferase specificfor the GalNAc acceptor moiety and the fucose donor moiety in thepresence of nucleotide-fucose. This process is illustrated on N-linkedglycans in FIG. 11 and Example 39.

[0572] In other exemplary embodiments, the peptide is expressed ininsect cells such as sf9 according to methods well known in the art.When a peptide having O-linked glycan consensus sites is expressed andsecreted from most sf9 cells, the majority of the O-linked glycans havethe structure, in the terms of Formula 2:

[0573] X²=H;

[0574] f=0 or 1;

[0575] n and m=0.

[0576] See, for example, Marchal et al., (2001, Biol. Chem.382:151-159). In one exemplary embodiment, the O-linked glycan on apeptide expressed in an insect cell is remodeled to a humanized glycanby contacting the glycans with a glycosyltransferase specific for aGalNAc acceptor molecule and a galactose donor molecule in the presenceof nucleotide-Gal; and then contacting the glycans with aglycosyltransferase specific for a Gal acceptor molecule and a SA donormolecule in the presence of nucleotide-SA. In another exemplaryembodiment, the O-linked glycans are remodeled further from thehumanized form to the sialylated Lewis X form by further contacting theglycans with a glycosyltransferase specific for a GalNAc acceptormolecule and a fucose donor molecule in the presence ofnucleotide-fucose.

[0577] In yet another exemplary embodiment, the peptide is expressed infungal cells, in particular S. cerevisiae cells, according to methodswell known in the art. When a peptide with O-linked glycans consensussites is expressed and secreted from S. cerevisiae cells, the majorityof the O-linked glycans have the structure:

[0578] |-AA-Man-Man₁₋₂.

[0579] See Gemmill and Trimble (1999, Biochim. Biophys. Acta1426:227-237). In order to remodel these O-linked glycans for use inhuman, it is preferable that the glycan be cleaved at the amino acidlevel and rebuilt from there.

[0580] In an exemplary embodiment, the glycan is the O-linked glycan ona peptide expressed in a fungal cell and is remodeled to a humanizedglycan by contacting the glycan with an endoglycosylase specific for anamino acid—GalNAc bond; and then contacting the glycan with aglycosyltransferase specific for a O-linked consensus site and a GalNAcdonor molecule in the presence of nucleotide-GalNAc; contacting theglycan with a glycosyltransferase specific for a GalNAc acceptormolecule and a galactose donor molecule in the presence ofnucleotide-Gal; and then contacting the glycans with aglycosyltransferase specific for a Gal acceptor molecule and a SA donormolecule in the presence of nucleotide-SA.

[0581] Alternately, in another exemplary embodiment, the glycan is theO-linked glycan on a peptide expressed in a fungal cell and is remodeledto a humanized glycan by contacting the glycan with an protein O-mannoseβ-1,2-N-acetylglucosaminyltransferase (POMGnTI) in the presence ofGlcNAc-nucleotide; then contacting the glycan with angalactosyltransferase in the presence of nucleotide-Gal; and thencontracting the glycan with an sialyltransferase in the presence ofnucleotide-SA.

[0582] In another exemplary embodiment, the peptide is expressed inbacterial cells, in particular E. coli cells, according to methods wellknown in the art. When a peptide with an O-linked glycan consensus siteis expressed in E. coli cells, the O-linked consensus site will not beglycosylated. In this case, the desired glycan molecule must be builtout from the peptide backbone in a manner similar to that describe forS. cerevisiae expression above. Further, when a peptide having anO-linked glycan is expressed in a eukaryotic cell without the properleader sequences to direct the nascent peptide to the golgi apparatus,the mature peptide is likely not to be glycosylated. In this case aswell, an O-linked glycosyl structure may be added to the peptide bybuilding out the glycan directly from the peptide O-linked consensussite. Further, when a protein is chemically modified with a sugarmoiety, it can also be remodeled as described herein.

[0583] These examples are meant to illustrate the invention, and not tolimit it in any way. One of skill in the art will appreciate that thesteps taken in each example may in some circumstances be performed in adifferent order to achieve the same result. One of skill in the art willalso understand that a different set of steps may also produce the sameresulting glycan. Futher, the preferred remodeled glycan is by no meansspecific to the expression system that the peptide is expressed in. Theremodeled glycans are only illustrative and one of skill in the art willknow how to take the principles from these examples and apply them topeptides produced in different expression systems to generate glycansnot specifically described herein.

[0584] C. Glycoconjugation, in General

[0585] The invention provides methods of preparing a conjugate of aglycosylated or an unglycosylated peptide. The conjugates of theinvention are formed between peptides and diverse species such aswater-soluble polymers, therapeutic moieties, diagnostic moieties,targeting moieties and the like. Also provided are conjugates thatinclude two or more peptides linked together through a linker arm, i.e.,multifunctional conjugates. The multi-functional conjugates of theinvention can include two or more copies of the same peptide or acollection of diverse peptides with different structures, and/orproperties.

[0586] The conjugates of the invention are formed by the enzymaticattachment of a modified sugar to the glycosylated or unglycosylatedpeptide. The modified sugar, when interposed between the peptide and themodifying group on the sugar becomes what is referred to herein as “anintact glycosyl linking group.” Using the exquisite selectivity ofenzymes, such as glycosyltransferases, the present method providespeptides that bear a desired group at one or more specific locations.Thus, according to the present invention, a modified sugar is attacheddirectly to a selected locus on the peptide chain or, alternatively, themodified sugar is appended onto a carbohydrate moiety of a peptide.Peptides in which modified sugars are linked to both a peptidecarbohydrate and directly to an amino acid residue of the peptidebackbone are also within the scope of the present invention.

[0587] In contrast to known chemical and enzymatic peptide elaborationstrategies, the methods of the invention make it possible to assemblepeptides and glycopeptides that have a substantially homogeneousderivatization pattern; the enzymes used in the invention are generallyselective for a particular amino acid residue or combination of aminoacid residues of the peptide or particular glycan structure. The methodsare also practical for large-scale production of modified peptides andglycopeptides. Thus, the methods of the invention provide a practicalmeans for large-scale preparation of peptides having preselectedsubstantially uniform derivatization patterns. The methods areparticularly well suited for modification of therapeutic peptides,including but not limited to, peptides that are incompletelyglycosylated during production in cell culture cells (e.g., mammaliancells, insect cells, plant cells, fungal cells, yeast cells, orprokaryotic cells) or transgenic plants or animals.

[0588] The methods of the invention also provide conjugates ofglycosylated and unglycosylated peptides with increased therapeutichalf-life due to, for example, reduced clearance rate, or reduced rateof uptake by the immune or reticuloendothelial system (RES). Moreover,the methods of the invention provide a means for masking antigenicdeterminants on peptides, thus reducing or eliminating a host immuneresponse against the peptide. Selective attachment of targeting agentscan also be used to target a peptide to a particular tissue or cellsurface receptor that is specific for the particular targeting agent.Moreover, there is provided a class of peptides that are specificallymodified with a therapeutic moiety.

[0589] 1. The Conjugates

[0590] In a first aspect, the present invention provides a conjugatebetween a peptide and a selected moiety. The link between the peptideand the selected moiety includes an intact glycosyl linking groupinterposed between the peptide and the selected moiety. As discussedherein, the selected moiety is essentially any species that can beattached to a saccharide unit, resulting in a “modified sugar” that isrecognized by an appropriate transferase enzyme, which appends themodified sugar onto the peptide. The saccharide component of themodified sugar, when interposed between the peptide and a selectedmoiety, becomes an “intact glycosyl linking group.” The glycosyl linkinggroup is formed from any mono- or oligo-saccharide that, aftermodification with a selected moiety, is a substrate for an appropriatetransferase.

[0591] The conjugates of the invention will typically correspond to thegeneral structure:

[0592] in which the symbols a, b, c, d and s represent a positive,non-zero integer; and t is either 0 or a positive integer. The “agent”is a therapeutic agent, a bioactive agent, a detectable label,water-soluble moiety or the like. The “agent” can be a peptide, e.g.,enzyme, antibody, antigen, etc. The linker can be any of a wide array oflinking groups, infra. Alternatively, the linker may be a single bond ora “zero order linker.” The identity of the peptide is withoutlimitation. Exemplary peptides are provided in FIG. 28.

[0593] In an exemplary embodiment, the selected moiety is awater-soluble polymer. The water-soluble polymer is covalently attachedto the peptide via an intact glycosyl linking group. The glycosyllinking group is covalently attached to either an amino acid residue ora glycosyl residue of the peptide. Alternatively, the glycosyl linkinggroup is attached to one or more glycosyl units of a glycopeptide. Theinvention also provides conjugates in which the glycosyl linking groupis attached to both an amino acid residue and a glycosyl residue.

[0594] In addition to providing conjugates that are formed through anenzymatically added intact glycosyl linking group, the present inventionprovides conjugates that are highly homogenous in their substitutionpatterns. Using the methods of the invention, it is possible to formpeptide conjugates in which essentially all of the modified sugarmoieties across a population of conjugates of the invention are attachedto multiple copies of a structurally identical amino acid or glycosylresidue. Thus, in a second aspect, the invention provides a peptideconjugate having a population of water-soluble polymer moieties, whichare covalently linked to the peptide through an intact glycosyl linkinggroup. In a preferred conjugate of the invention, essentially eachmember of the population is linked via the glycosyl linking group to aglycosyl residue of the peptide, and each glycosyl residue of thepeptide to which the glycosyl linking group is attached has the samestructure.

[0595] Also provided is a peptide conjugate having a population ofwater-soluble polymer moieties covalently linked thereto through anintact glycosyl linking group. In a preferred embodiment, essentiallyevery member of the population of water soluble polymer moieties islinked to an amino acid residue of the peptide via an intact glycosyllinking group, and each amino acid residue having an intact glycosyllinking group attached thereto has the same structure.

[0596] The present invention also provides conjugates analogous to thosedescribed above in which the peptide is conjugated to a therapeuticmoiety, diagnostic moiety, targeting moiety, toxin moiety or the likevia an intact glycosyl linking group. Each of the above-recited moietiescan be a small molecule, natural polymer (e.g., peptide) or syntheticpolymer.

[0597] In an exemplary embodiment, interleukin-2 (IL-2) is conjugated totransferrin via a bifunctional linker that includes an intact glycosyllinking group at each terminus of the PEG moiety (Scheme 1). Forexample, one terminus of the PEG linker is functionalized with an intactsialic acid linker that is attached to transferrin and the other isfunctionalized with an intact GalNAc linker that is attached to IL-2.

[0598] In another exemplary embodiment, EPO is conjugated totransferrin. In another exemplary embodiment, EPO is conjugated to glialderived neurotropic growth factor (GDNF). In these embodiments, eachconjugation is accomplished via a bifunctional linker that includes anintact glycosyl linking group at each terminus of the PEG moiety, asaforementioned. Transferrin transfers the protein across the blood brainbarrier.

[0599] As set forth in the Figures appended hereto, the conjugates ofthe invention can include intact glycosyl linking groups that are mono-or multi-valent (e.g., antennary structures), see, FIGS. 14-22. Theconjugates of the invention also include glycosyl linking groups thatare O-linked glycans originating from serine or threonine (FIG. 11).Thus, conjugates of the invention include both species in which aselected moiety is attached to a peptide via a monovalent glycosyllinking group. Also included within the invention are conjugates inwhich more than one selected moiety is attached to a peptide via amultivalent linking group. One or more proteins can be conjugatedtogether to take advantage of their biophysical and biologicalproperties.

[0600] In a still further embodiment, the invention provides conjugatesthat localize selectively in a particular tissue due to the presence ofa targeting agent as a component of the conjugate. In an exemplaryembodiment, the targeting agent is a protein. Exemplary proteins includetransferrin (brain, blood pool), human serum (HS)-glycoprotein (bone,brain, blood pool), antibodies (brain, tissue with antibody-specificantigen, blood pool), coagulation Factors V-XII (damaged tissue, clots,cancer, blood pool), serum proteins, e.g., α-acid glycoprotein, fetuin,α-fetal protein (brain, blood pool), β2-glycoprotein (liver,atherosclerosis plaques, brain, blood pool), G-CSF, GM-CSF, M-CSF, andEPO (immune stimulation, cancers, blood pool, red blood celloverproduction, neuroprotection), and albumin (increase in half-life).

[0601] In addition to the conjugates discussed above, the presentinvention provides methods for preparing these and other conjugates.Thus, in a further aspect, the invention provides a method of forming acovalent conjugate between a selected moiety and a peptide.Additionally, the invention provides methods for targeting conjugates ofthe invention to a particular tissue or region of the body.

[0602] In exemplary embodiments, the conjugate is formed between awater-soluble polymer, a therapeutic moiety, targeting moiety or abiomolecule, and a glycosylated or non-glycosylated peptide. Thepolymer, therapeutic moiety or biomolecule is conjugated to the peptidevia an intact glycosyl linking group, which is interposed between, andcovalently linked to both the peptide and the modifying group (e.g.,water-soluble polymer). The method includes contacting the peptide witha mixture containing a modified sugar and a glycosyltransferase forwhich the modified sugar is a substrate. The reaction is conducted underconditions sufficient to form a covalent bond between the modified sugarand the peptide. The sugar moiety of the modified sugar is preferablyselected from nucleotide sugars, activated sugars and sugars, which areneither nucleotides nor activated.

[0603] In one embodiment, the invention provides a method for linkingtwo or more peptides through a linking group. The linking group is ofany useful structure and may be selected from straight-chain andbranched chain structures. Preferably, each terminus of the linker,which is attached to a peptide, includes a modified sugar (i.e., anascent intact glycosyl linking group).

[0604] In an exemplary method of the invention, two peptides are linkedtogether via a linker moiety that includes a PEG linker. The constructconforms to the general structure set forth in the cartoon above. Asdescribed herein, the construct of the invention includes two intactglycosyl linking groups (i.e., s+t=1). The focus on a PEG linker thatincludes two glycosyl groups is for purposes of clarity and should notbe interpreted as limiting the identity of linker arms of use in thisembodiment of the invention.

[0605] Thus, a PEG moiety is functionalized at a first terminus with afirst glycosyl unit and at a second terminus with a second glycosylunit. The first and second glycosyl units are preferably substrates fordifferent transferases, allowing orthogonal attachment of the first andsecond peptides to the first and second glycosyl units, respectively. Inpractice, the (glycosyl)¹-PEG-(glycosyl)² linker is contacted with thefirst peptide and a first transferase for which the first glycosyl unitis a substrate, thereby forming (peptide)¹-(glycosyl)¹-PEG-(glycosyl)².The first transferase and/or unreacted peptide is then optionallyremoved from the reaction mixture. The second peptide and a secondtransferase for which the second glycosyl unit is a substrate are addedto the (peptide)¹-(glycosyl)¹-PEG-(glycosyl)² conjugate, forming(peptide)¹-(glycosyl)¹-PEG-(glycosyl)²-(peptide)². Those of skill in theart will appreciate that the method outlined above is also applicable toforming conjugates between more than two peptides by, for example, theuse of a branched PEG, dendrimer, poly(amino acid), polysaccharide orthe like.

[0606] As noted previously, in an exemplary embodiment, interleukin-2(IL-2) is conjugated to transferrin via a bifunctional linker thatincludes an intact glycosyl linking group at each terminus of the PEGmoiety (Scheme 1). The IL-2 conjugate has an in vivo half-life that isincreased over that of IL-2 alone by virtue of the greater molecularsize of the conjugate. Moreover, the conjugation of IL-2 to transferrinserves to selectively target the conjugate to the brain. For example,one terminus of the PEG linker is functionalized with a CMP-sialic acidand the other is functionalized with an UDP-GalNAc. The linker iscombined with IL-2 in the presence of a GalNAc transferase, resulting inthe attachment of the GalNAc of the linker arm to a serine and/orthreonine residue on the IL-2.

[0607] In another exemplary embodiment, transferrin is conjugated to anucleic acid for use in gene therapy.

[0608] The processes described above can be carried through as manycycles as desired, and is not limited to forming a conjugate between twopeptides with a single linker. Moreover, those of skill in the art willappreciate that the reactions functionalizing the intact glycosyllinking groups at the termini of the PEG (or other) linker with thepeptide can occur simultaneously in the same reaction vessel, or theycan be carried out in a step-wise fashion. When the reactions arecarried out in a step-wise manner, the conjugate produced at each stepis optionally purified from one or more reaction components (e.g.,enzymes, peptides).

[0609] A still further exemplary embodiment is set forth in Scheme 2.Scheme 2 shows a method of preparing a conjugate that targets a selectedprotein, e.g., EPO, to bone and increases the circulatory half-life ofthe selected protein.

[0610] The use of reactive derivatives of PEG (or other linkers) toattach one or more peptide moieties to the linker is within the scope ofthe present invention. The invention is not limited by the identity ofthe reactive PEG analogue. Many activated derivatives of poly(ethyleneglycol) are available commercially and in the literature. It is wellwithin the abilities of one of skill to choose, and synthesize ifnecessary, an appropriate activated PEG derivative with which to preparea substrate useful in the present invention. See, Abuchowski et al.Cancer Biochem. Biophys., 7: 175-186 (1984); Abuchowski et al., J. Biol.Chem., 252: 3582-3586 (1977); Jackson et al., Anal. Biochem., 165:114-127 (1987); Koide et al., Biochem Biophys. Res. Commun., 111:659-667 (1983)), tresylate (Nilsson et al., Methods Enzymol., 104: 56-69(1984); Delgado et al., Biotechnol. Appl. Biochem., 12: 119-128 (1990));N-hydroxysuccinimide derived active esters (Buckmann et al., Makromol.Chem., 182: 1379-1384 (1981); Joppich et al., Makromol. Chem., 180:1381-1384 (1979); Abuchowski et al., Cancer Biochem. Biophys., 7:175-186 (1984); Katreet al. Proc. Natl. Acad. Sci. U.S.A., 84: 1487-1491(1987); Kitamura et al., Cancer Res., 51: 4310-4315 (1991); Boccu etal., Z. Naturforsch., 38C: 94-99 (1983), carbonates (Zalipsky et al.,POLY(ETHYLENE GLYCOL) CHEMISTRY: BIOTECHNICAL AND BIOMEDICALAPPLICATIONS, Harris, Ed., Plenum Press, New York, 1992, pp. 347-370;Zalipsky et al., Biotechnol. Appl. Biochem., 15: 100-114 (1992);Veronese et al., Appl. Biochem. Biotech., 11: 141-152 (1985)),imidazolyl formates (Beauchamp et al., Anal. Biochem., 131: 25-33(1983); Berger et al., Blood, 71: 1641-1647 (1988)), 4-dithiopyridines(Woghiren et al., Bioconjugate Chem., 4: 314-318 (1993)), isocyanates(Byun et al., ASAIO Journal, M649-M-653 (1992)) and epoxides (U.S. Pat.No. 4,806,595, issued to Noishiki et al., (1989). Other linking groupsinclude the urethane linkage between amino groups and activated PEG.See, Veronese, et al., Appl. Biochem. Biotechnol., 11: 141-152 (1985).

[0611] In another exemplary embodiment in which a reactive PEGderivative is utilized, the invention provides a method for extendingthe blood-circulation half-life of a selected peptide, in essencetargeting the peptide to the blood pool, by conjugating the peptide to asynthetic or natural polymer of a size sufficient to retard thefiltration of the protein by the glomerulus (e.g., albumin). Thisembodiment of the invention is illustrated in Scheme 3 in whicherythropoietin (EPO) is conjugated to albumin via a PEG linker using acombination of chemical and enzymatic modification.

[0612] Thus, as shown in Scheme 3, an amino acid residue of albumin ismodified with a reactive PEG derivative, such as X-PEG-(CMP-sialicacid), in which X is an activating group (e.g., active ester,isothiocyanate, etc). The PEG derivative and EPO are combined andcontacted with a transferase for which CMP-sialic acid is a substrate.In a further illustrative embodiment, an ε-amine of lysine is reactedwith the N-hydroxysuccinimide ester of the PEG-linker to form thealbumin conjugate. The CMP-sialic acid of the linker is enzymaticallyconjugated to an appropriate residue on EPO, e.g., Gal, thereby formingthe conjugate. Those of skill will appreciate that the above-describedmethod is not limited to the reaction partners set forth. Moreover, themethod can be practiced to form conjugates that include more than twoprotein moieties by, for example, utilizing a branched linker havingmore than two termini.

[0613] 2. Modified Sugars

[0614] Modified glycosyl donor species (“modified sugars”) arepreferably selected from modified sugar nucleotides, activated modifiedsugars and modified sugars that are simple saccharides that are neithernucleotides nor activated. Any desired carbohydrate structure can beadded to a peptide using the methods of the invention. Typically, thestructure will be a monosaccharide, but the present invention is notlimited to the use of modified monosaccharide sugars; oligosaccharidesand polysaccharides are useful as well.

[0615] The modifying group is attached to a sugar moiety by enzymaticmeans, chemical means or a combination thereof, thereby producing amodified sugar. The sugars are substituted at any position that allowsfor the attachment of the modifying moiety, yet which still allows thesugar to function as a substrate for the enzyme used to ligate themodified sugar to the peptide. In a preferred embodiment, when sialicacid is the sugar, the sialic acid is substituted with the modifyinggroup at either the 9-position on the pyruvyl side chain or at the5-position on the amine moiety that is normally acetylated in sialicacid.

[0616] In certain embodiments of the present invention, a modified sugarnucleotide is utilized to add the modified sugar to the peptide.Exemplary sugar nucleotides that are used in the present invention intheir modified form include nucleotide mono-, di- or triphosphates oranalogs thereof. In a preferred embodiment, the modified sugarnucleotide is selected from a UDP-glycoside, CMP-glycoside, or aGDP-glycoside. Even more preferably, the modified sugar nucleotide isselected from an UDP-galactose, UDP-galactosamine, UDP-glucose,UDP-glucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid, or CMP-NeuAc.N-acetylamine derivatives of the sugar nucleotides are also of use inthe method of the invention.

[0617] The invention also provides methods for synthesizing a modifiedpeptide using a modified sugar, e.g., modified-galactose, -fucose, and-sialic acid. When a modified sialic acid is used, either asialyltransferase or a trans-sialidase (for α2,3-linked sialic acidonly) can be used in these methods.

[0618] In other embodiments, the modified sugar is an activated sugar.Activated modified sugars, which are useful in the present invention aretypically glycosides which have been synthetically altered to include anactivated leaving group. As used herein, the term “activated leavinggroup” refers to those moieties, which are easily displaced inenzyme-regulated nucleophilic substitution reactions. Many activatedsugars are known in the art. See, for example, Vocadlo et al., InCARBOHYDRATE CHEMISTRY AND BIOLOGY, Vol. 2, Ernst et al. Ed., Wiley-VCHVerlag: Weinheim, Germany, 2000; Kodama et al., Tetrahedron Lett. 34:6419 (1993); Lougheed, et al., J. Biol. Chem. 274: 37717 (1999)).

[0619] Examples of activating groups (leaving groups) include fluoro,chloro, bromo, tosylate ester, mesylate ester, triflate ester and thelike. Preferred activated leaving groups, for use in the presentinvention, are those that do not significantly sterically encumber theenzymatic transfer of the glycoside to the acceptor. Accordingly,preferred embodiments of activated glycoside derivatives includeglycosyl fluorides and glycosyl mesylates, with glycosyl fluorides beingparticularly preferred. Among the glycosyl fluorides, α-galactosylfluoride, α-mannosyl fluoride, α-glucosyl fluoride, α-fucosyl fluoride,α-xylosyl fluoride, α-sialyl fluoride, α-N-acetylglucosaminyl fluoride,α-N-acetylgalactosaminyl fluoride, β-galactosyl fluoride, β-mannosylfluoride, β-glucosyl fluoride, β-fucosyl fluoride, β-xylosyl fluoride,β-sialyl fluoride, α-N-acetylglucosaminyl fluoride andα-N-acetylgalactosaminyl fluoride are most preferred.

[0620] By way of illustration, glycosyl fluorides can be prepared fromthe free sugar by first acetylating the sugar and then treating it withHF/pyridine. This generates the thermodynamically most stable anomer ofthe protected (acetylated) glycosyl fluoride (i.e., the α-glycosylfluoride). If the less stable anomer (i.e., the β-glycosyl fluoride) isdesired, it can be prepared by converting the peracetylated sugar withHBr/HOAc or with HCl to generate the anomeric bromide or chloride. Thisintermediate is reacted with a fluoride salt such as silver fluoride togenerate the glycosyl fluoride. Acetylated glycosyl fluorides may bedeprotected by reaction with mild (catalytic) base in methanol (e.g.NaOMe/MeOH). In addition, many glycosyl fluorides are commerciallyavailable.

[0621] Other activated glycosyl derivatives can be prepared usingconventional methods known to those of skill in the art. For example,glycosyl mesylates can be prepared by treatment of the fully benzylatedhemiacetal form of the sugar with mesyl chloride, followed by catalytichydrogenation to remove the benzyl groups.

[0622] In a further exemplary embodiment, the modified sugar is anoligosaccharide having an antennary structure. In a preferredembodiment, one or more of the termini of the antennae bear themodifying moiety. When more than one modifying moiety is attached to anoligosaccharide having an antennary structure, the oligosaccharide isuseful to “amplify” the modifying moiety; each oligosaccharide unitconjugated to the peptide attaches multiple copies of the modifyinggroup to the peptide. The general structure of a typical chelate of theinvention as set forth in the drawing above, encompasses multivalentspecies resulting from preparing a conjugate of the invention utilizingan antennary structure. Many antennary saccharide structures are knownin the art, and the present method can be practiced with them withoutlimitation.

[0623] Exemplary modifying groups are discussed below. The modifyinggroups can be selected for one or more desirable property. Exemplaryproperties include, but are not limited to, enhanced pharmacokinetics,enhanced pharmacodynamics, improved biodistribution, providing apolyvalent species, improved water solubility, enhanced or diminishedlipophilicity, and tissue targeting.

[0624] D. Peptide Conjugates

[0625] a) Water-Soluble Polymers

[0626] The hydrophilicity of a selected peptide is enhanced byconjugation with polar molecules such as amine-, ester-, hydroxyl- andpolyhydroxyl-containing molecules. Representative examples include, butare not limited to, polylysine, polyethyleneimine, poly(ethylene glycol)and poly(propyleneglycol). Preferred water-soluble polymers areessentially non-fluorescent, or emit such a minimal amount offluorescence that they are inappropriate for use as a fluorescent markerin an assay. Polymers that are not naturally occurring sugars may beused. In addition, the use of an otherwise naturally occurring sugarthat is modified by covalent attachment of another entity (e.g.,poly(ethylene glycol), poly(propylene glycol), poly(aspartate),biomolecule, therapeutic moiety, diagnostic moiety, etc.) is alsocontemplated. In another exemplary embodiment, a therapeutic sugarmoiety is conjugated to a linker arm and the sugar-linker arm issubsequently conjugated to a peptide via a method of the invention.

[0627] Methods and chemistry for activation of water-soluble polymersand saccharides as well as methods for conjugating saccharides andpolymers to various species are described in the literature. Commonlyused methods for activation of polymers include activation of functionalgroups with cyanogen bromide, periodate, glutaraldehyde, biepoxides,epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halides,trichlorotriazine, etc. (see, R. F. Taylor, (1991), PROTEINIMMOBILISATION. FUNDAMENTALS AND APPLICATIONS, Marcel Dekker, N.Y.; S.S. Wong, (1992), CHEMISTRY OF PROTEIN CONJUGATION AND CROSSLINKING, CRCPress, Boca Raton; G. T. Hermanson et al., (1993), IMMOBILIZED AFFINITYLIGAND TECHNIQUES, Academic Press, N.Y.; Dunn, R. L., et al., Eds.POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium Series Vol.469, American Chemical Society, Washington, D.C. 1991).

[0628] Routes for preparing reactive PEG molecules and formingconjugates using the reactive molecules are known in the art. Forexample, U.S. Pat. No. 5,672,662 discloses a water soluble andisolatable conjugate of an active ester of a polymer acid selected fromlinear or branched poly(alkylene oxides), poly(oxyethylated polyols),poly(olefinic alcohols), and poly(acrylomorpholine), wherein the polymerhas about 44 or more recurring units.

[0629] U.S. Pat. No. 6,376,604 sets forth a method for preparing awater-soluble 1-benzotriazolylcarbonate ester of a water-soluble andnon-peptidic polymer by reacting a terminal hydroxyl of the polymer withdi(1-benzotriazoyl)carbonate in an organic solvent. The active ester isused to form conjugates with a biologically active agent such as aprotein or peptide.

[0630] WO 99/45964 describes a conjugate comprising a biologicallyactive agent and an activated water soluble polymer comprising a polymerbackbone having at least one terminus linked to the polymer backbonethrough a stable linkage, wherein at least one terminus comprises abranching moiety having proximal reactive groups linked to the branchingmoiety, in which the biologically active agent is linked to at least oneof the proximal reactive groups. Other branched poly(ethylene glycols)are described in WO 96/21469, U.S. Pat. No. 5,932,462 describes aconjugate formed with a branched PEG molecule that includes a branchedterminus that includes reactive functional groups. The free reactivegroups are available to react with a biologically active species, suchas a protein or peptide, forming conjugates between the poly(ethyleneglycol) and the biologically active species. U.S. Pat. No. 5,446,090describes a bifunctional PEG linker and its use in forming conjugateshaving a peptide at each of the PEG linker termini.

[0631] Conjugates that include degradable PEG linkages are described inWO 99/34833; and WO 99/14259, as well as in U.S. Pat. No. 6,348,558.Such degradable linkages are applicable in the present invention.

[0632] Although both reactive PEG derivatives and conjugates formedusing the derivatives are known in the art, until the present invention,it was not recognized that a conjugate could be formed between PEG (orother polymer) and another species, such as a peptide or glycopeptide,through an intact glycosyl linking group.

[0633] Many water-soluble polymers are known to those of skill in theart and are useful in practicing the present invention. The termwater-soluble polymer encompasses species such as saccharides (e.g.,dextran, amylose, hyalouronic acid, poly(sialic acid), heparans,heparins, etc.); poly (amino acids), e.g., poly(glutamic acid); nucleicacids; synthetic polymers (e.g., poly(acrylic acid), poly(ethers), e.g.,poly(ethylene glycol); peptides, proteins, and the like. The presentinvention may be practiced with any water-soluble polymer with the solelimitation that the polymer must include a point at which the remainderof the conjugate can be attached.

[0634] Methods for activation of polymers can also be found in WO94/17039, U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat.No. 5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No.5,281,698, and more WO 93/15189, and for conjugation between activatedpolymers and peptides, e.g. Coagulation Factor VIII (WO 94/15625),hemoglobin (WO 94/09027), oxygen carrying molecule (U.S. Pat. No.4,412,989), ribonuclease and superoxide dismutase (Veronese at al., App.Biochem. Biotech. 11: 141-45 (1985)).

[0635] Preferred water-soluble polymers are those in which a substantialproportion of the polymer molecules in a sample of the polymer are ofapproximately the same molecular weight; such polymers are“homodisperse.”

[0636] The present invention is further illustrated by reference to apoly(ethylene glycol) conjugate. Several reviews and monographs on thefunctionalization and conjugation of PEG are available. See, forexample, Harris, Macronol. Chem. Phys. C25: 325-373 (1985); Scouten,Methods in Enzymology 135: 30-65 (1987); Wong et al., Enzyme Microb.Technol. 14: 866-874 (1992); Delgado et al., Critical Reviews inTherapeutic Drug Carrier Systems 9: 249-304 (1992); Zalipsky,Bioconjugate Chem. 6: 150-165 (1995); and Bhadra, et al., Pharmazie,57:5-29 (2002).

[0637] Poly(ethylene glycol) molecules suitable for use in the inventioninclude, but are not limited to, those described by the followingFormula 3:

[0638] R═H, alkyl, benzyl, aryl, acetal, OHC—, H₂N—CH₂CH₂—, HS—CH₂CH₂—,

[0639]  , -sugar-nucleotide, protein, methyl, ethyl;

[0640] X, Y, W, U (independently selected)=O, S, NH, N—R′;

[0641] R′, R′″ (independently selected)=alkyl, benzyl, aryl, alkyl aryl,pyridyl, substituted aryl, arylalkyl, acylaryl;

[0642] n=1 to 2000;

[0643] m, q, p (independently selected)=0 to 20

[0644] o=0 to 20;

[0645] Z=HO, NH₂, halogen, S—R′″, activated esters,

[0646]  -sugar-nucleotide, protein, imidazole, HOBT, tetrazole, halide;and V=HO, NH₂, halogen, S—R′″, activated esters, activated amides,-sugar-nucleotide, protein.

[0647] In preferred embodiments, the poly(ethylene glycol) molecule isselected from the following:

[0648] The poly(ethylene glycol) useful in forming the conjugate of theinvention is either linear or branched. Branched poly(ethylene glycol)molecules suitable for use in the invention include, but are not limitedto, those described by the following Formula:

[0649] R′, R″, R′″ (independently selected)=H, alkyl, benzyl, aryl,acetal, OHC—, H₂N—CH₂CH₂—, HS—CH²CH₂—, —(CH₂)_(q)CY-Z,-sugar-nucleotide, protein, methyl, ethyl, heteroaryl, acylalkyl,acylaryl, acylalkylaryl;

[0650] X, Y, W, A, B (independently selected)=O, S, NH, N—R′, (CH₂)₁;

[0651] n, p (independently selected)=1 to 2000;

[0652] m, q, o (independently selected)=0 to 20;

[0653] Z=HO, NH₂, halogen, S—R′″, activated esters,

[0654]  -sugar-nucleotide, protein;

[0655] V=HO, NH₂, halogen, S—R′″, activated esters, activated amides,

[0656] -sugar-nucleotide, protein.

[0657] The in vivo half-life, area under the curve, and/or residencetime of therapeutic peptides can also be enhanced with water-solublepolymers such as polyethylene glycol (PEG) and polypropylene glycol(PPG). For example, chemical modification of proteins with PEG(PEGylation) increases their molecular size and decreases their surface-and functional group-accessibility, each of which are dependent on thesize of the PEG attached to the protein. This results in an improvementof plasma half-lives and in proteolytic-stability, and a decrease inimmunogenicity and hepatic uptake (Chaffee et al. J. Clin. Invest. 89:1643-1651 (1992); Pyatak et al. Res. Commun. Chem. Pathol Pharmacol. 29:113-127 (1980)). PEGylation of interleukin-2 has been reported toincrease its antitumor potency in vivo (Katre et al. Proc. Natl. Acad.Sci. USA. 84: 1487-1491 (1987)) and PEGylation of a F(ab′)2 derived fromthe monoclonal antibody A7 has improved its tumor localization (Kitamuraet al. Biochem. Biophys. Res. Commun. 28: 1387-1394 (1990)).

[0658] In one preferred embodiment, the in vivo half-life of a peptidederivatized with a water-soluble polymer by a method of the invention isincreased relevant to the in vivo half-life of the non-derivatizedpeptide. In another preferred embodiment, the area under the curve of apeptide derivatized with a water-soluble polymer using a method of theinvention is increased relevant to the area under the curve of thenon-derivatized peptide. In another preferred embodiment, the residencetime of a peptide derivatized with a water-soluble polymer using amethod of the invention is increased relevant to the residence time ofthe non-derivatized peptide. Techniques to determine the in vivohalf-life, the area under the curve and the residence time are wellknown in the art. Descriptions of such techniques can be found in J. G.Wagner, 1993, Pharmacokinetics for the Pharmaceutical Scientist,Technomic Publishing Company, Inc. Lancaster Pa.

[0659] The increase in peptide in vivo half-life is best expressed as arange of percent increase in this quantity. The lower end of the rangeof percent increase is about 40%, about 60%, about 80%, about 100%,about 150% or about 200%. The upper end of the range is about 60%, about80%, about 100%, about 150%, or more than about 250%.

[0660] In an exemplary embodiment, the present invention provides aPEGylated follicle stimulating hormone (Examples 23 and 24). In afurther exemplary embodiment, the invention provides a PEGylatedtransferrin (Example 42).

[0661] Other exemplary water-soluble polymers of use in the inventioninclude, but are not limited to linear or branched poly(alkyleneoxides), poly(oxyethylated polyols), poly(olefinic alcohols), andpoly(acrylomorpholine), dextran, starch, poly(amino acids), etc.

[0662] b) Water-Insoluble Polymers

[0663] The conjugates of the invention may also include one or morewater-insoluble polymers. This embodiment of the invention isillustrated by the use of the conjugate as a vehicle with which todeliver a therapeutic peptide in a controlled manner. Polymeric drugdelivery systems are known in the art. See, for example, Dunn et al.,Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium SeriesVol. 469, American Chemical Society, Washington, D.C. 1991. Those ofskill in the art will appreciate that substantially any known drugdelivery system is applicable to the conjugates of the presentinvention.

[0664] Representative water-insoluble polymers include, but are notlimited to, polyphosphazines, poly(vinyl alcohols), polyamides,polycarbonates, polyalkylenes, polyacrylamides, polyalkylene glycols,polyalkylene oxides, polyalkylene terephthalates, polyvinyl ethers,polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone,polyglycolides, polysiloxanes, polyurethanes, poly(methyl methacrylate),poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutylmethacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate),poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methylacrylate), poly(isopropyl acrylate), poly(isobutyl acrylate),poly(octadecyl acrylate) polyethylene, polypropylene, poly(ethyleneglycol), poly(ethylene oxide), poly (ethylene terephthalate), poly(vinylacetate), polyvinyl chloride, polystyrene, polyvinyl pyrrolidone,pluronics and polyvinylphenol and copolymers thereof.

[0665] Synthetically modified natural polymers of use in conjugates ofthe invention include, but are not limited to, alkyl celluloses,hydroxyalkyl celluloses, cellulose ethers, cellulose esters, andnitrocelluloses. Particularly preferred members of the broad classes ofsynthetically modified natural polymers include, but are not limited to,methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, celluloseacetate, cellulose propionate, cellulose acetate butyrate, celluloseacetate phthalate, carboxymethyl cellulose, cellulose triacetate,cellulose sulfate sodium salt, and polymers of acrylic and methacrylicesters and alginic acid.

[0666] These and the other polymers discussed herein can be readilyobtained from commercial sources such as Sigma Chemical Co. (St. Louis,Mo.), Polysciences (Warrenton, Pa.), Aldrich (Milwaukee, Wis.), Fluka(Ronkonkoma, N.Y.), and BioRad (Richmond, Calif.), or else synthesizedfrom monomers obtained from these suppliers using standard techniques.

[0667] Representative biodegradable polymers of use in the conjugates ofthe invention include, but are not limited to, polylactides,polyglycolides and copolymers thereof, poly(ethylene terephthalate),poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone),poly(lactide-co-glycolide), polyanhydrides, polyorthoesters, blends andcopolymers thereof. Of particular use are compositions that form gels,such as those including collagen, pluronics and the like.

[0668] The polymers of use in the invention include “hybrid’ polymersthat include water-insoluble materials having within at least a portionof their structure, a bioresorbable molecule. An example of such apolymer is one that includes a water-insoluble copolymer, which has abioresorbable region, a hydrophilic region and a plurality ofcrosslinkable functional groups per polymer chain.

[0669] For purposes of the present invention, “water-insolublematerials” includes materials that are substantially insoluble in wateror water-containing environments. Thus, although certain regions orsegments of the copolymer may be hydrophilic or even water-soluble, thepolymer molecule, as a whole, does not to any substantial measuredissolve in water.

[0670] For purposes of the present invention, the term “bioresorbablemolecule” includes a region that is capable of being metabolized orbroken down and resorbed and/or eliminated through normal excretoryroutes by the body. Such metabolites or break down products arepreferably substantially non-toxic to the body.

[0671] The bioresorbable region may be either hydrophobic orhydrophilic, so long as the copolymer composition as a whole is notrendered water-soluble. Thus, the bioresorbable region is selected basedon the preference that the polymer, as a whole, remains water-insoluble.Accordingly, the relative properties, i.e., the kinds of functionalgroups contained by, and the relative proportions of the bioresorbableregion, and the hydrophilic region are selected to ensure that usefulbioresorbable compositions remain water-insoluble.

[0672] Exemplary resorbable polymers include, for example, syntheticallyproduced resorbable block copolymers of poly(α-hydroxy-carboxylicacid)/poly(oxyalkylene, (see, Cohn et al., U.S. Pat. No. 4,826,945).These copolymers are not crosslinked and are water-soluble so that thebody can excrete the degraded block copolymer compositions. See, Youneset al., J. Biomed. Mater. Res. 21: 1301-1316 (1987); and Cohn et al., J.Biomed. Mater. Res. 22: 993-1009 (1988).

[0673] Presently preferred bioresorbable polymers include one or morecomponents selected from poly(esters), poly(hydroxy acids),poly(lactones), poly(amides), poly(ester-amides), poly (amino acids),poly(anhydrides), poly(orthoesters), poly(carbonates),poly(phosphazines), poly(phosphoesters), poly(thioesters),polysaccharides and mixtures thereof. More preferably still, thebiosresorbable polymer includes a poly(hydroxy) acid component. Of thepoly(hydroxy) acids, polylactic acid, polyglycolic acid, polycaproicacid, polybutyric acid, polyvaleric acid and copolymers and mixturesthereof are preferred.

[0674] In addition to forming fragments that are absorbed in vivo(“bioresorbed”), preferred polymeric coatings for use in the methods ofthe invention can also form an excretable and/or metabolizable fragment.

[0675] Higher order copolymers can also be used in the presentinvention. For example, Casey et al., U.S. Pat. No. 4,438,253, whichissued on Mar. 20, 1984, discloses tri-block copolymers produced fromthe transesterification of poly(glycolic acid) and an hydroxyl-endedpoly(alkylene glycol). Such compositions are disclosed for use asresorbable monofilament sutures. The flexibility of such compositions iscontrolled by the incorporation of an aromatic orthocarbonate, such astetra-p-tolyl orthocarbonate into the copolymer structure.

[0676] Other coatings based on lactic and/or glycolic acids can also beutilized. For example, Spinu, U.S. Pat. No. 5,202,413, which issued onApr. 13, 1993, discloses biodegradable multi-block copolymers havingsequentially ordered blocks of polylactide and/or polyglycolide producedby ring-opening polymerization of lactide and/or glycolide onto eitheran oligomeric diol or a diamine residue followed by chain extension witha di-functional compound, such as, a diisocyanate, diacylchloride ordichlorosilane.

[0677] Bioresorbable regions of coatings useful in the present inventioncan be designed to be hydrolytically and/or enzymatically cleavable. Forpurposes of the present invention, “hydrolytically cleavable” refers tothe susceptibility of the copolymer, especially the bioresorbableregion, to hydrolysis in water or a water-containing environment.Similarly, “enzymatically cleavable” as used herein refers to thesusceptibility of the copolymer, especially the bioresorbable region, tocleavage by endogenous or exogenous enzymes.

[0678] When placed within the body, the hydrophilic region can beprocessed into excretable and/or metabolizable fragments. Thus, thehydrophilic region can include, for example, polyethers, polyalkyleneoxides, polyols, poly(vinyl pyrrolidine), poly(vinyl alcohol),poly(alkyl oxazolines), polysaccharides, carbohydrates, peptides,proteins and copolymers and mixtures thereof. Furthermore, thehydrophilic region can also be, for example, a poly(alkylene) oxide.Such poly(alkylene) oxides can include, for example, poly(ethylene)oxide, poly(propylene) oxide and mixtures and copolymers thereof.

[0679] Polymers that are components of hydrogels are also useful in thepresent invention. Hydrogels are polymeric materials that are capable ofabsorbing relatively large quantities of water. Examples of hydrogelforming compounds include, but are not limited to, polyacrylic acids,sodium carboxymethylcellulose, polyvinyl alcohol, polyvinyl pyrrolidine,gelatin, carrageenan and other polysaccharides,hydroxyethylenemethacrylic acid (HEMA), as well as derivatives thereof,and the like. Hydrogels can be produced that are stable, biodegradableand bioresorbable. Moreover, hydrogel compositions can include subunitsthat exhibit one or more of these properties.

[0680] Bio-compatible hydrogel compositions whose integrity can becontrolled through crosslinking are known and are presently preferredfor use in the methods of the invention. For example, Hubbell et al.,U.S. Pat. No. 5,410,016, which issued on Apr. 25, 1995 and U.S. Pat. No.5,529,914, which issued on Jun. 25, 1996, disclose water-solublesystems, which are crosslinked block copolymers having a water-solublecentral block segment sandwiched between two hydrolytically labileextensions. Such copolymers are further end-capped withphotopolymerizable acrylate functionalities. When crosslinked, thesesystems become hydrogels. The water soluble central block of suchcopolymers can include poly(ethylene glycol); whereas, thehydrolytically labile extensions can be a poly(α-hydroxy acid), such aspolyglycolic acid or polylactic acid. See, Sawhney et al.,Macromolecules 26: 581-587 (1993).

[0681] In another preferred embodiment, the gel is a thermoreversiblegel. Thermoreversible gels including components, such as pluronics,collagen, gelatin, hyalouronic acid, polysaccharides, polyurethanehydrogel, polyurethane-urea hydrogel and combinations thereof arepresently preferred.

[0682] In yet another exemplary embodiment, the conjugate of theinvention includes a component of a liposome. Liposomes can be preparedaccording to methods known to those skilled in the art, for example, asdescribed in Eppstein et al., U.S. Pat. No. 4,522,811, which issued onJun. 11, 1985. For example, liposome formulations may be prepared bydissolving appropriate lipid(s) (such as stearoyl phosphatidylethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidylcholine, and cholesterol) in an inorganic solvent that is thenevaporated, leaving behind a thin film of dried lipid on the surface ofthe container. An aqueous solution of the active compound or itspharmaceutically acceptable salt is then introduced into the container.The container is then swirled by hand to free lipid material from thesides of the container and to disperse lipid aggregates, thereby formingthe liposomal suspension.

[0683] The above-recited microparticles and methods of preparing themicroparticles are offered by way of example and they are not intendedto define the scope of microparticles of use in the present invention.It will be apparent to those of skill in the art that an array ofmicroparticles, fabricated by different methods, are of use in thepresent invention.

[0684] c) Biomolecules

[0685] In another preferred embodiment, the modified sugar bears abiomolecule. In still further preferred embodiments, the biomolecule isa functional protein, enzyme, antigen, antibody, peptide, nucleic acid(e.g., single nucleotides or nucleosides, oligonucleotides,polynucleotides and single- and higher-stranded nucleic acids), lectin,receptor or a combination thereof.

[0686] Some preferred biomolecules are essentially non-fluorescent, oremit such a minimal amount of fluorescence that they are inappropriatefor use as a fluorescent marker in an assay. Other biomolecules may befluorescent. The use of an otherwise naturally occurring sugar that ismodified by covalent attachment of another entity (e.g., PEG,biomolecule, therapeutic moiety, diagnostic moiety, etc.) isappropriate. In an exemplary embodiment, a sugar moiety, which is abiomolecule, is conjugated to a linker arm and the sugar-linker armcassette is subsequently conjugated to a peptide via a method of theinvention.

[0687] Biomolecules useful in practicing the present invention can bederived from any source. The biomolecules can be isolated from naturalsources or they can be produced by synthetic methods. Peptides can benatural peptides or mutated peptides. Mutations can be effected bychemical mutagenesis, site-directed mutagenesis or other means ofinducing mutations known to those of skill in the art. Peptides usefulin practicing the instant invention include, for example, enzymes,antigens, antibodies and receptors. Antibodies can be either polyclonalor monoclonal; either intact or fragments. The peptides are optionallythe products of a program of directed evolution.

[0688] Both naturally derived and synthetic peptides and nucleic acidsare of use in conjunction with the present invention; these moleculescan be attached to a sugar residue component or a crosslinking agent byany available reactive group. For example, peptides can be attachedthrough a reactive amine, carboxyl, sulfhydryl, or hydroxyl group. Thereactive group can reside at a peptide terminus or at a site internal tothe peptide chain. Nucleic acids can be attached through a reactivegroup on a base (e.g., exocyclic amine) or an available hydroxyl groupon a sugar moiety (e.g., 3′- or 5′-hydroxyl). The peptide and nucleicacid chains can be further derivatized at one or more sites to allow forthe attachment of appropriate reactive groups onto the chain. See,Chrisey et al. Nucleic Acids Res. 24: 3031-3039 (1996).

[0689] In a further preferred embodiment, the biomolecule is selected todirect the peptide modified by the methods of the invention to aspecific tissue, thereby enhancing the delivery of the peptide to thattissue relative to the amount of underivatized peptide that is deliveredto the tissue. In a still further preferred embodiment, the amount ofderivatized peptide delivered to a specific tissue within a selectedtime period is enhanced by derivatization by at least about 20%, morepreferably, at least about 40%, and more preferably still, at leastabout 100%. Presently, preferred biomolecules for targeting applicationsinclude antibodies, hormones and ligands for cell-surface receptors.Exemplary targeting biomolecules include, but are not limited to, anantibody specific for the transferrin receptor for delivery of themolecule to the brain (Penichet et al., 1999, J. Immunol. 163:4421-4426;Pardridge, 2002, Adv. Exp. Med. Biol. 513:397-430), a peptide thatrecognizes the vasculature of the prostate (Arap et al., 2002, PNAS99:1527-1531), and an antibody specific for lung caveolae (McIntosh etal., 2002, PNAS 99:1996-2001).

[0690] In a presently preferred embodiment, the modifying group is aprotein. In an exemplary embodiment, the protein is an interferon. Theinterferons are antiviral glycoproteins that, in humans, are secreted byhuman primary fibroblasts after induction with virus or double-strandedRNA. Interferons are of interest as therapeutics, e.g., antivirals andtreatment of multiple sclerosis. For references discussing interferon-β,see, e.g., Yu, et al., J. Neuroimmunol., 64(1):91-100 (1996); Schmidt,J., J. Neurosci. Res., 65(1):59-67 (2001); Wender, et al., FoliaNeuropathol., 39(2):91-93 (2001); Martin, et al., Springer Semin.Immunopathol., 18(1):1-24 (1996); Takane, et al., J. Pharmacol. Exp.Ther., 294(2):746-752 (2000); Sburlati, et al., Biotechnol. Prog.,14:189-192 (1998); Dodd, et al., Biochimica et Biophysica Acta,787:183-187 (1984); Edelbaum, et al., J. Interferon Res., 12:449-453(1992); Conradt, et al., J. Biol. Chem., 262(30):14600-14605 (1987);Civas, et al., Eur. J. Biochem., 173:311-316 (1988); Demolder, et al.,J. Biotechnol., 32:179-189 (1994); Sedmak, et al., J. Interferon Res.,9(Suppl 1):S61-S65 (1989); Kagawa, et al., J. Biol. Chem.,263(33):17508-17515 (1988); Hershenson, et al., U.S. Pat. No. 4,894,330;Jayaram, et al., J. Interferon Res., 3(2):177-180 (1983); Menge, et al.,Develop. Biol. Standard., 66:391-401 (1987); Vonk, et al., J. InterferonRes., 3(2):169-175 (1983); and Adolf, et al., J. Interferon Res.,10:255-267 (1990). For references relevant to interferon-(x, see, Asano,et al., Eur. J. Cancer, 27(Suppl 4):S21-S25 (1991); Nagy, et al.,Anticancer Research, 8(3):467-470 (1988); Dron, et al., J. Biol. Regul.Homeost. Agents, 3(1):13-19 (1989); Habib, et al., Am. Surg.,67(3):257-260 (3/2001); and Sugyiama, et al., Eur. J. Biochem.,217:921-927 (1993).

[0691] In an exemplary interferon conjugate, interferon P is conjugatedto a second peptide via a linker arm. The linker arm includes an intactglycosyl linking group through which it is attached to the secondpeptide via a method of the invention. The linker arm also optionallyincludes a second intact glycosyl linking group, through which it isattached to the interferon.

[0692] In another exemplary embodiment, the invention provides aconjugate of follicle stimulating hormone (FSH). FSH is a glycoproteinhormone. See, for example, Saneyoshi, et al., Biol. Reprod.,65:1686-1690 (2001); Hakola, et al., J. Endocrinol., 158:441-448 (1998);Stanton, et al., Mol. Cell. Endocrinol., 125:133-141 (1996); Walton, etal., J. Clin. Endocrinol. Metab., 86(8):3675-3685 (08/2001);Ulloa-Aguirre, et al., Endocrine, 11(3):205-215 (12/1999);Castro-Fernández, et al. I, J. Clin. Endocrinol. Matab.,85(12):4603-4610 (2000); Prevost, Rebecca R., Pharmacotherapy,18(5):1001-1010 (1998); Linskens, et al., The FASEB Journal, 13:639-645(04/1999); Butnev, et al., Biol. Reprod., 58:458-469 (1998); Muyan, etal., Mol. Endo., 12(5):766-772 (1998); Min, et al., Endo. J.,43(5):585-593 (1996); Boime, et al., Recent Progress in HormoneResearch, 34:271-289 (1999); and Rafferty, et al., J. Endo., 145:527-533(1995). The FSH conjugate can be formed in a manner similar to thatdescribed for interferon.

[0693] In yet another exemplary embodiment, the conjugate includeserythropoietin (EPO). EPO is known to mediate response to hypoxia and tostimulate the production of red blood cells. For pertinent references,see, Cerami, et al., Seminars in Oncology, 28(2)(Suppl 8):66-70(04/2001). An exemplary EPO conjugate is formed analogously to theconjugate of interferon.

[0694] In a further exemplary embodiment, the invention provides aconjugate of human granulocyte colony stimulating factor (G-CSF). G-CSFis a glycoprotein that stimulates proliferation, differentiation andactivation of neutropoietic progenitor cells into functionally matureneutrophils. Injected G-CSF is known to be rapidly cleared from thebody. See, for example, Nohynek, et al., Cancer Chemother. Pharmacol.,39:259-266 (1997); Lord, et al., Clinical Cancer Research,7(7):2085-2090 (07/2001); Rotondaro, et al., Molecular Biotechnology,11(2):117-128 (1999); and Bonig, et al., Bone Marrow Transplantation,28:259-264 (2001). An exemplary conjugate of G-CSF is prepared asdiscussed above for the conjugate of the interferons. One of skill inthe art will appreciate that many other proteins may be conjugated tointerferon using the methods and compositions of the invention,including but not limited to, the peptides listed in Tables 7 and 8(presented elsewhere herein) and FIG. 28, and in FIGS. 29-57, whereindividual modification schemes are presented.

[0695] In still a further exemplary embodiment, there is provided aconjugate with biotin. Thus, for example, a selectively biotinylatedpeptide is elaborated by the attachment of an avidin or streptavidinmoiety bearing one or more modifying groups.

[0696] In a further preferred embodiment, the biomolecule is selected todirect the peptide modified by the methods of the invention to aspecific intracellular compartment, thereby enhancing the delivery ofthe peptide to that intracellular compartment relative to the amount ofunderivatized peptide that is delivered to the tissue. In a stillfurther preferred embodiment, the amount of derivatized peptidedelivered to a specific intracellular compartment within a selected timeperiod is enhanced by derivatization by at least about 20%, morepreferably, at least about 40%, and more preferably still, at leastabout 100%. In another particularly preferred embodiment, thebiomolecule is linked to the peptide by a cleavable linker that canhydrolyze once internalized. Presently, preferred biomolecules forintracellular targeting applications include transferrin,lactotransferrin (lactoferrin), melanotransferrin (p97), ceruloplasmin,and divalent cation transporter, as well as antibodies directed againstspecific vascular targets. Contemplated linkages include, but are notlimited to, protein-sugar-linker-sugar-protein,protein-sugar-linker-protein and multivalent forms thereof, andprotein-sugar-linker-drug where the drug includes small molecules,peptides, lipids, among others.

[0697] Site-specific and target-oriented delivery of therapeutic agentsis desirable for the purpose of treating a wide variety of humandiseases, such as different types of malignancies and certainneurological disorders. Such procedures are accompanied by fewer sideeffects and a higher efficiacy of drug. Various principles have beenrelied on in designing these delivery systems. For a review, seeGarnett, Advanced Drug Delivery Reviews 53:171-216 (2001).

[0698] One important consideration in designing a drug delivery systemto target tissues specifically. The discovery of tumor surface antigenshas made it possible to develop therapeutic approaches where tumor cellsdisplaying definable surface antigens are specifically targeted andkilled. There are three main classes of therapeutic monoclonalantibodies (antibody) that have demonstrated effectiveness in humanclinical trials in treating malignancies: (1) unconjugated MAb, whicheither directly induces growth inhibition and/or apoptosis, orindirectly activates host defense mechanisms to mediate antitumorcytotoxicity; (2) drug-conjugated MAb, which preferentially delivers apotent cytotoxic toxin to the tumor cells and therefore minimizes thesystemic cytotoxicity commonly associated with conventionalchemotherapy; and (3) radioisotope-conjugated MAb, which delivers asterilizing dose of radiation to the tumor. See review by Reff et al.,Cancer Control 9:152-166 (2002).

[0699] In order to arm MAbs with the power to kill malignant cells, theMAbs can be connected to a toxin, which may be obtained from a plant,bacterial, or fungal source, to form chimeric proteins calledimmunotoxins. Frequently used plant toxins are divided into two classes:(1) holotoxins (or class II ribosome inactivating proteins), such asricin, abrin, mistletoe lectin, and modeccin, and (2) hemitoxins (classI ribosome inactivating proteins), such as pokeweed antiviral protein(PAP), saporin, Bryodin 1, bouganin, and gelonin. Commonly usedbacterial toxins include diphtheria toxin (DT) and Pseudomonas exotoxin(PE). Kreitman, Current Pharmaceutical Biotechnology 2:313-325 (2001).Other toxins contemplated for use with the present invention include,but are not limited to, those in Table 2. TABLE 2 Toxins. ChemicalStructure Toxin Name/ Source/ CAS RN/ Indication/ Activity (IC50 nM);Alternate ID Analogs Toxicity Mechanism Tumor Type

SW-163E/ 260794-24-9; Cancer and not reported 0.3 P388 Streptomyces spSNA 260794-25-0/ Antibacterial/ 0.2 A2780 15896/ SW-163C; low toxicity(mice ip) 0.4 KB SW-163E SW-163A; 1.6 colon SW-163B 1.3 HL-60

Thiocoraline/ 173046-02-1 Breast Cancer; DNA lung, colon, CNSMicromonospora marina Melanoma; Non-small Polymerase melanoma(actinomycete) lung cancer/ alpha not reported inhibitor (blocks cellprogression from G1 to S)

Trunkasmide A¹/ 181758-83-8 Cancer/ not reported cell culture (IC50 inLissoclinum sp (aascidian) not reported micrograms/mL); 0.5 P388; 0.5A549; 0.5 HT-29; 1.0 MEL-28

Palauamine²/ 148717-58-2 Lung cancer/ not reported cell culture(IC50 inStylotella agminata LD50 (i.p. in mice) is 13 micrograms/mL); (sponge)mg/Kg 0.1 P388 0.2 A549 (lung) 2 HT-29 (colon) 10 KB

Halichondrin B/ 103614-76-2/ cancer/ antitubulin; NCI tumor panel;Halichondria Okadai, isohomohalic myelotoxicity dose cell cycle GI(50)from 50 nM to Axinell Carteri and hondrin B limiting (dogs, rats)inhibitor 0.1 nM; Phankell carteri (inhibits LC50's from 40 μM to(sponges)/ GTP binding 0.1 nM (many 0.1 to 25 NSC-609385 to tubulin) nM)

Isohomo-halichondrin B/ 157078-48-3/ melanoma, lung, CNS, antitubulin;IC50's in 0.1 nM range Halichondria Okadai, halichondrin colon, ovary/cell cycle (NCI tumor panel) Axinell Carteri and B not reportedinhibitor Phankell carteri (inhibits (sponges)/ GTP binding NSC-650467to tubulin)

Halichondrin B analogs/ 253128-15-3/ solid tumors/ tubulin cell culture(not semi-synthetic starting ER-076349; not reported binding reported);from Halichondria ER-086526; agent; animal models active Okadai, AxinellCarteri B-1793; disruption of (tumor regression and Phankell carteriE-7389 mitotic observed) in lymphoma, (sponges)/ spindles colon(multi-drug ER-076349; ER-086526; resistant). B-1793; E-7389

NK-130119/ 132707-68-7 antifungal and not reported 25 ng/mL colonStreptomyces anticancer/ 8.5 ng/mL lung bottropensis/ not reportedNK-130119

Tetrocarcin A/ 73666-84-9/ cancer/ inhibits the not reported notreported/ analogs are not reported anti- KF-67544 reported apoptoticfunctino of Bcl2

Gilvusmycin/ 195052-09-6 cancer/ not reported IC50's in ng/mL;Streptomyces QM16 not reported 0.08 P388 0.86 K562 (CML) 0.72 A431 (EC)0.75 MKN28 (GI); (for all <1 nM)

IB-96212/ 220858-11-7/ Cancer and not reported IC50's in ng/mL: marineactinomycete/ IB-96212; Antibacterial/ 0.1 P388 IB-96212 IB-98214; notreported IB-97227

BE-56384³/ 207570-04-5 cancer/ not reported IC50's in ng/mL:Streptomyces Sp./ not reported 0.1 P388 BE-56384 0.29 colon 26 34 DLD-10.12 PC-13 0.12 MKM-45

Palmitoylrhizoxin/ 135819-69-1/ cancer/ tubulin not reportedsemi-synthetic; Rhizopus Analog of binds LDL; less binding chinensisrhizoxin cytotoxic than rhizoxin agent (cell cycle inhibitor)

Rhizoxin/ 95917-95-6; melanoma, lung, CNS, tubulin NCI tumor panel (NSCRhizopus chinensis/ 90996-54-6 colon, ovary, renal, binding 332598);WF-1360; NSC-332598; breast, head and neck/ agent (cell log GI50's;FR-900216 Rapid Drug clearance; cycle 50 nM to 50 fM: High AUCcorrelates inhibitor) log LC50's: with high toxicity 50 μM to 0.5 nM(several cell lines at 50 fM).

Dolastatin-10/ 110417-88-4/ prostate, melanoma, tubulin NCI tumor panelDolabella auricularia (sea other leukemia/ binding (60 cell line; GI50);hare)/ Dolistatins myelotoxicity (at greater (tubulin 25 nM to 1 pM(most <1 NSC-376128 (ie. 15) and than 0.3 pM) aggregation) nM) (threecell lines analogs μM)

soblidotin/ 149606-27-9/ cancer (pancreas, tubulin cell culture: colon,synthetic/ analogs esophageal colon, breast, binding melanoma, M5076TZT-1027; auristatin PE prepared lung, etc)/ agent tumors, P388 with75-85% MTD was 1.8 mg/Kg inhibition (dose (IV); toxicity not notreported) reported

Dolastatin-15/ not reported/ cancer/ Tubulin NCI tumor panel (60Dolabella auricularia (sea other not reported binding cell line; GI50);25 hare) Dolistatins (tubuline nM to 39 pM (most <1 (ie. 15) andaggregation) nM) (one cell line 2.5 analogs μM); most active in breast

Cemadotin⁴/ 1159776-69-9/ melanoma/ tubulin NCI tumor panel (NCSSynthetic; Parent many analogs hypertension, myocardial bindingD-669356); active in Dolastatin-15 was isolated ischemia and (tubulinbreast, ovary, from Dolabella myelosuppression were aggregation)endometrial, sarcomas auricularia (sea hare)/ dose-limiting toxicities.and drug resistant cell LU-103793; NSC lines. Data not public. D-669356

Epothilone A/ not reported/ cancer/ tubulin IC 50's of; Synthetic orisolated from many analogs not reported binding 1.5 nM MCF-7 (breast)Sorangium cellulosum (tubulin 27.1 nM MCF-7/ADR (myxococcales) strainpolymeriza- 2.1 nM KB-31 SO ce90) ion) (melanoma) 3.2 nM HCT-116

Epothilone B/ 152044054-7/ Solid tumors (breast, tubulin IC50's of;Synthetic or isolated from many analogs ovarian, etc)/ binding 0.18 nMMCF-7 Sporangium cellulosum well tolerated; t½ of (tubulin (breast)(myxococcales) strain So 2.5 hrs; partial polymeriza- 2.92 nM MCF-7/ADRce90)/ responses (phase I); tion) 0.19 nM KB-31 EPO-906 diarrhea majorside (melanoma) effect. 0.42 nM HCT-116; broad activity reported

Epothilone Analog/ not reported/ cancer/ tubulin IC50's of 0.30 toSynthetic or semi- hundreds of not reported binding 1.80 nM in varioussynthetic; Original lead, analogs (tubulin tumor cell lines; EpothioneA, isolated polymeriza- active in drug resistant from Sorangium tion)cell lines cellulosum (myxococcales) strain So ce90)/ ZK-EPO

Epothilone D/ 189452-10-9/ Solid tumors (breast, tubulin NCI tumor panel(NSC- Epothilone D, isolated many analogs ovarian, etc)/ binding 703147;IC50); from Sorangium emesis and anemia; t½ (tubulin 0.19 nM KB-31cellulosum of 5-10 hrs. polymeriza- (melanoma) (myxococcales) strain Sotion) 0.42 nM HCT-116; ce90)/ broad activity reported KOS-862 StructureNot Identified Epothilone D analog⁵/ 189453-10-9/ Solid tumors; tubulinnot reported Synthetic or semi- hundreds of not reported bindingsynthetic; Original lead, analogs (tubulin Epothilone D, isolatedpolymeriza- from Sorangium tion) cellulosum (myxococcales) strain Soce90)/ KOS-166-24

Epothilone Analog/ not reported/ cancer; tubulin not reported Synthetic;Original lead, hundreds of not reported binding Epothilone A, isolatedanalogs (tubulin from Sorangium polymeriza- cellulosum tion)(myxococcales) strain So ce90)/ CGP-85715

Epothilone Analog/ 219989-84-1/ non-small cell Lung, tubulin NCI tumorPanel (NSC- Synthetic or semi- hundreds of breast, stomach tumor binding710428 & NSC- synthetic; Original lead, analogs (objective responses in(tubulin 710468); 8-32 nM Epothilone B, isolated breast ovarian andlung)/ polymeriza- (NCI data not available) from Sorangium severtoxicity (fatigue, tion) cellulosum anorexia, nauseas, (myxococcales)strain So vomiting, neuropathy ce90)/ myalgia) BMS-247550

Epothilone Analog/ not reported/ advanced cancers/ tubulin broadactivity with Synthetic or semi- hundreds of adverse events (diarrhea,binding IC50's of 0.7 to 10 nM synthetic; Original lead, analogs nausea,vomiting, (tubulin Epothilone B, isolated fatigue, neutropenia);polymeriza- from Sorangium t½ of 3.5 hrs; tion) cellulosum improvedwater (myxococcales) strain So solubility to BMS ce90)/ 247550.BMS-310705

Discodermolide/ 127943-53-7/ solid tumors/ tubulin Broad activity (A549-synthetic; orginally analogs less not reported; 100-fold stabilizingnsclung, prostate, P388, isolated from Discodermia potent increase inwater agent ovarian with IC50's dissoluta (deep water solubility overtaxol (similar to about 10 nM) including sponge); rare compound taxol)multi-drug resistant cell (7 mg per 0.5 Kg sponge/ lines; XAA-296

Chondramide D/ 172430-63-6 cancer/ tubulin 5 nM A-549 not reported notreported binding (epidermoid carcinoma) agent; actin 15 nM A-498(kidney) polymeriza- 14 nM A549 (lung) tion inhibitor 5 nM SK-OV-3(ovary) 3 nM U-937 (lymphoma)

Cryptophycin analogs 204990-60-3 solid tumors, colon tubulin broadactivity (lung, (including 52, 55 and and 186256- cancer/ polymeriza-breast, colon, leukemia) others)⁶/ 67-7/ Phase II studies halted tioninhibitor with IC50's of 2 to 40 Nostoc sp GSV 224 (blue- many potentbecause of severe pM; active against green algae) isolated analogstoxicity with one death multi-drug resistance Cryptophycin l./ preparedat resulting from drug; cell lines (resistant to LY-355703; Ly-355702;Lilly MDR pump). NCI NSC-667642 tumor panel, GI50's from 100 nM to 10pM; LC50's from 100 nM to 25 pM.

Cryptophycin 8/ 168482-36-8; solid tumors/ tubulin broad spectrumsemi-synthetic; starting 168482-40-4; not reported polymeriza-anticancer activity (cell material from Nostoc sp. 18665-94-1; tioninhibitor culture) including 124689-65-2; multi-drug resistant125546-14-7/ tumors cryptophycin 5, 15 and 35

Cryptophycin analogs⁷/ 219660-54-5/ solid tumors/ topoisomer- notreported synthetic; semi-synthetic, LY-404292 not reported aseinhibitors starting material from Nostoc sp./ LY-404291

Arenastatin A analogs⁸/ not reported/ cancer/ inhibits 8.7 nM (5 pg/mL)KB Dysidea arenaria (marine analogs not reported tubulin(nasopharyngeal); NCI sponge)/ prepared polymeriza- tumor panel(GI50's); Cryptophycin B; NSC- tion 100 pM to 3 pM 670038

Phomopsin A/ not reported Liver cancer (not as tubulin potent anticancerDiaporte toxicus or potent in other cancers)/ binding activityespecially Phomopsin not reported agent against liver cancerleptostromiformis (fungi)

Curacin A and analogs/ 155233-30-0/ Cancer/ Tubulin broad activity(cancer Lyngbya majuscula (blue analogs have not reported binding celllines); 1-29 nM green cyanobacterium) been prepared agent

Hemiasterlins A & B not reported/ Cancer/ Antimitotic broad activity;and analogs⁹/ criamide A & not reported agent 0.3-3 nM MCF7 Cymbastelasp. B; (tubulin (breast); geodiamiolid- binding 0.4 ng/mL P388 G agent)

Spongistatins (1-9)¹⁰/ 149715-96-8; cancer/ tubulin Most potentcompounds Spirastrell spinispirulifera 158734-18-0; not reported bindingever tested in NCI panel (sea sponge) 158681-42-6; agent cell line (meanGI50's 158080-65-0; of 0.1 nM; 150642-07-2; Spongistatin-1 GI50's153698-80-7; of 0.025-0.035 nM with 153745-94-9; extremely potent150624-44-5; activity against a subset 158734-19-1/ of highly otherchemoresistant tumor spongistatins types

Maytansine/ 35846-53-8/ cancer/ tubulin Broad Activity in NCI Maytenussp./ other related severe toxocity binding tumor panel (NSC- NSC-153858macrolids agent (causes 153858; NSC-153858); extensive NCI tumor panel,disassembly GI50's from 3 μM to of the 0.1 pM; LC50's from microtubule250 μM to 10 pM. Two and totally different experiments prevents gavevery different tubulin potencies. spiralization)

Maytansine-IgG(EGFR not reported/ breast, head and neck, EGFR notreported directed)-conjugate¹¹/ other related Squamous cell binding andsemi-synthetic; starting macrolides carcinoma/ tubulin material fromMaytenus not reported binding sp.

Maytansine-IgG(CD56 not reported/ Neuroendocrine, small- CD56antigen-specific antigen)-conjugate¹², 3.5 other related cell lung,carcinoma/ binding and cytotoxicity (cell drug molecules per IgG/macrolides mild toxocity (fatigue, tubulin culture; epidermal,semi-synthetic; starting nausea, headaches and binding breast, renalovarian material from Maytenus mild peripheral colon) with IC50's ofsp./ neuropathy); no 10-40 pM; animal huN901-DM1 heamtological toxicity;studies (miceSCLC MTD 60 mg/Kg. I.V., tumor-alone and in weekly for 4weeks; only combination with taxol stable disease reported or cisplatincompletely (humans) eliminated tumors).

Maytansine-IgG(CEA not reported/ non-small-cell lung, DEA bindingantigen-specific antigen)-conjugate¹³, 4 other related carcinomapancreas, and tubulin cytotoxicity (cell drug molecules per IgG/macrolides lung, colon/ binding culture; epidermal, semi-synthetic;starting mild toxocity (fatigue, breast, renal ovarian material fromMaytenus nausea, headaches and colon) with IC50's of sp./ mildperipheral 10-40 pM; animal C424-DM1 neuropathy); pancreatic studies(mice: lipase elevated; MTD 88 melanoma [COLO- mg/Kg, I.V., every 21205]-alone and in days; only stable disease combination with taxolreported (humans); t½ or cisplatin completely was 44 hr. eliminatedtumors);

Geldanamycin/ 30562-34-6/ cancer/ binds Hsp 90 NCI tumor panel (cellStreptomyces natural not reported chaperone culture); 5.3 to 100hygroscopicus var. derivatives and inhibits nM; most active in Geldanus/function colon, lung and NSC-212518; Antibiotic leukemia. NCI tumor U29135; NSC-122750 panel, GI50's from 10 μM to 0.1 nM; LC50's from 100 μMto 100 nM. Two assays with very different potencies.

Geldanamycin Analog/ 745747-14-7/ solid tumors/ binds Hsp 90 cellculture (not semi-synthetic;/ Kosan, NCI Dose limiting toxicitieschaperone reported); animal CP-127374; 17-AAG; and UK (anemia, anorexia,and inhibits models active (tumor NSC-330507 looking for diarrhea,nausea and function regression observed) in analogs with vomiting); t½(i.v.) is breast, ovary, longer t½ about 90 min; no melanoma, colon. andoral objective responses activity; measured at 88 mg/Kg analogs (i.v.daily for 5 days, include: NSC- every 21 days); 255110; 682300; 683661;683663.

Geldanamycin analog/ not reported/ solid tumors/ binds Hsp 90 notreported semi-synthetic;/ analogs not reported chaperone CP-202567prepared and inhibits function

Geldanamycin 345232-44-2/ breast/ binds Hsp 90 cell culture (noconjugates/ analogs not reported chaperone reported); animalsemi-synthetic;/ prepared and inhibits models performed LY-294002-GM;function; PI3K-1-GM binds and inhibits PI-3 kinase Structure NotReported Geldanamycin Analog/ not reported/ breast, prostate/ binds Hsp90 not reported not reported/ analogs not reported chaperone CNF-101prepared and inhibits function Structure Not Reported Geldanamycin- notreported/ prostate/ binds Hsp 90 not reported; conjugate testosteroneconjugate/ analogs not reported chaperone has a 15-fold selectivesemi-synthetic/ prepared and inhibits cytotoxicity for GMT-1 functionand androgen positive testosterone prostate cells receptors where it isinternalized

Podophyllotoxin/ 518-28-5/ Verruca vulgaris, tubulin broad activity(cell Podophyllum sp. many analogs Condyloma/ inhibitor and culture)with IC50's in severe toxicity when topoisomer- μM range given i.v. ors.c. ase inhibitor

esperamicin-A1/ 99674-26-7 cancer/ DNA highly potent activity not known/not reported (suspected cleaving (cell culture); animal BBM-1675A1;severe toxicity) agent models highly potent BMY-28175; with optimal doseof GGM-1675 0.16 micrograms/Kg

C-1027¹⁴/ 120177-69-7 cancer (examined DNA extremely potent (cellStreptomyces setonii hepatoma, breast, lung cleaving culture) IC50's inpM C-1027/ and leukemia/ agent and fM; conjugated to C-1027 not reportedantibodies the potency remains the same (ie. 5.5 to 42 pM);

Calicheamicin- 113440-58-7; AML/ CNA Kills CD33+ cells (HL-60, IgG(CD33antigen)- 220578-59-6/ mild toxicity cleaving 60, NOMO-1, andconjugate¹⁵/ several agent NKM-1) at 100 ng/mL; semi-synthetic: reportedin MDR cell lines are not Micromonospora patents effected by the drug.echinosporal gemtuzumab ozogamicin; mylotarg; WAY-CMA-676; CMA-676;CDP-771

Calicheamicin-IgG- 113440-58-7; cancer/ DNA TBD conjugates¹⁶/220578-59-6 not reported cleaving semi-synthetic: agent Micromonosporaechinospora

Calicheamicin- not reported cancer/ DNA all human cancer; data IgG(OBA1antigen) not reported cleaving not reported conjugate/ agentsemi-synthetic: Micromonospora echinosporal OBA1-H8

Calicheamicin- not reported non-Hodgkin lymphoma, DNA all human cancer;data IgG(CD22 antigen) cancer/ cleaving not reported conjugate/ notreported agent semi-synthetic: Micromonospora echinospora/ CMC-544parially esterified polystyrene maleic acid copolymer (SMA) conjugatedto neocarzinostatin (NCS) Neocarzinostatin¹⁷/ 123760-07-6; liver cancerand brain DNA cell culture data not semi-synthetic; 9014-02-2 cancer/cleaving reported. Streptomyces not reported agent carconistaticus/Zinostatin stimalamer; YM-881; YM-16881 IgG (TES-23)-conjugated toneocarzinostatin Neocarzinostatin/ not reported solid tumors/ DNA cellculture data not not reported/ toxocity not reported; the cleavingreported. TES-23-NCS TES-23 antibody aent and (without anticancerimmunostim- agent) was as effective at ulator eliminating tumors as thedrug conjugated protein

Kedarcidin¹⁸/ 128512-40-3; cancer/ DNA cell culture (IC50's inStreptoalloteichus sp 128512-39-0/ not reported cleaving ng/mL), 0.4HCT116; NOV strain L5856, ATCC chromophore agent 0.3 HCT116/VP35; 53650/and protein 0.3 HCT116/VM46; NSC-646276 conjugate 0.2 A2780; 1.3A2780/DDP. animal models in P388 and B-16 melanoma. NCI tumor panel,GI50's from 50 μM to 5 μM.

Eleutherobins/ 174545-76-7/ cancer/ tubulin similar potency to taxol;marine coral sarcodictyins not reported binding not effective against(marine coral) agent MDR cell lines

Bryostatin-1/ 83314-01-6 leukemia, melanoma, immunostim- not reportedBugula neritina (marine lung, cancer/ ulant (TNF, bryosoan)/ myalgia;accumulated GMCSF, GMY-45618; NSC-339555 toxicity; poor water etc);solubility; dose limiting enhances cell toxicity kill by currentanticancer agents

FR-901228/ 128517-07-7 leukemia, T-cell histone In vitro cell lines (NCIChromobacterium lymphoma, cancer/ deacetylase tumor panel); violaceumstrain 968/ toxic doses (LD50) 6.4 inhibitor IC50's of between 0.56NSC-63-176; FK-228 and 10 mg/Kg, ip and iv and 4.1 nM (breast,respectively; GI lung, gastric colon, toxicity, lymphoid leukemia)atrophy; dose limiting toxicity (human) 18 mg/Kg/ t½ of 8 hrs (human)

Chlamydocin/ 53342-16-8 cancer/ histone not reported (cell not reportednot reported deacetylase culture); inhibitor inhibits histonedeacetylase at an IC50 of 1.3 nM

Phorboxazole A¹⁹/ 181377-57-1; leukemia, myeloma/ not reported NCI tumorpanel marine sponge 165689-31-6; not reported (induces (details notreported); 180911-82-4; apoptosis) IC50's of 1-10 nM. The 165883-76-1/inhibition values analogs (clonogenic growth of prepared human cancercells) at 10 nM ranged from 6.2 to >99.9% against NALM-6 human B-lineage acute lymophobastic leukemia cells, BT-20 breast cancer cellsand U373 gliobastoma cells, with the specified compound showinginhibition values in the range of 42.4 to >99.9% against these celllines.; IC50's are nM for MDR cell lines.

Apicularen A/ 220757-06-2/ cancer/ not reported IC50's of 0.1 to 3Chondromyces robustus natural not reported ng/mL (KB-3-A, KB-derivatives Va, K562, HL60, U937, A498, A549, PV3 and SK-OV3)

Taxol/ 33069624/ cancer; breast, prostate, tubulin NCI tumor panel;Pacific yew and fungi/ many analogs ovary, colon, lung, head bindingGI50's of 3 nM to 1 Paclitaxel; NSC-125973 & neck, etc./ agent μM;severe toxicity (grade III TGI 50 nM to 25 μM and IV)

Vitilevuamide/ 191681-63-7 cancer/ tubulin cell culture; IC50's ofDidemnum cuculiferum not reported binding 6-311 nM (panel of orPolysyncraton agent tumor cell lines lithostrotum HCT116 cells, A549cells, SK-MEL-5 cells A498 cells). The increase in lifespan (ILS) forCDF1 mice after ip injection of P388 tumor cells was in the range of −45to +70% over the dose range of 0.13 to 0.006 mg/kg.

Didemnin B/ 77327-05-0; non-Hodgkin's inhibits NCI 60-tumor panelTrididemnum solidum/ 77327-04-9; lymphoma, breast, protein (GI50's): 100nM to 50 NSC-2325319; INC 77327-06-1/ carcinoma, CNS, colon/ synthesisvia fM. 24505 other related Discontinued due to EF-1 Not potent againstnatural cardiotoxicity; nausea, MDR cell lines. products neuro-musculartoxicity and vomiting MTD 6.3 mg/Kg; toxicity prevented achieving aclinically signif. effect; rapidly cleared (t½ 4.8 hrs

Leptomycin B/ 87081-35-4 NCI 60-tumor panel Streptomyces sp. strain(GI50's): ATS 1287/ 8 μM to 1 pM; (LC50): NSC-364372; elactocin 250 μMto 10 nM (several cell lines at 0.1 nM). Two testing results with verydifferent potencies.

Cryptopleurin/ NCI 60-tumor panel not known/ (GI50's): 19 nM to 1NSC-19912 pM; (LC50): 40 μM to 10 nM (several cell lines at 1 pM).

Silicicolin/ 19186-35-7 NCI60-tumor panel not known/ (GI50's): ˜100 nMto 3 NSC-403148, nM; (LC50): 50 μM to deoxypodophyllotoxin, 10 nMdesoxypodophyllotoxin podophyllotoxin, deoxysilicicolin

Scillaren A/ 124-99-2 NCI 60-tumor panel not known/ (GI50's): 50 nM to0.1 NSC-7525; Gluco- nM; proscillaridin A; (LC50): 250 μM to 0.1Scillaren A nM

Cinerubin A-HCl/ not reported NCI 60-tumor panel not known/ (GI50's): 15nM to 10 NSC-243022; Cinerubin pM; (LC50): 100 μM A hydrochloride; to 6nM CL 86-F2 HCl; CL-86-F2-hydrochloride

[0700] Conventional immunotoxins contain an MAb chemically conjugated toa toxin that is mutated or chemically modified to minimized binding tonormal cells. Examples include anti-B4-blocked ricin, targeting CD5; andRFB4-deglycosylated ricin A chain, targeting CD22. Recombinantimmunotoxins developed more recently are chimeric proteins consisting ofthe variable region of an antibody directed against a tumor antigenfused to a protein toxin using recombinant DNA technology. The toxin isalso frequently genetically modified to remove normal tissue bindingsites but retain its cytotoxicity. A large number of differentiationantigens, overexpressed receptors, or cancer-specific antigens have beenidentified as targets for immunotoxins, e.g., CD19, CD22, CD20, IL-2receptor (CD25), CD33, IL-4 receptor, EGF receptor and its mutants,ErB2, Lewis carbohydrate, mesothelin, transferrin receptor, GM-CSFreceptor, Ras, Bcr-Abl, and c-Kit, for the treatment of a variety ofmalignancies including hematopoietic cancers, glioma, and breast, colon,ovarian, bladder, and gastrointestinal cancers. See e.g., Brinkmann etal., Expert Opin. Biol. Ther. 1:693-702 (2001); Perentesis and Sievers,Hematology/Oncology Clinics of North America 15:677-701 (2001).

[0701] MAbs conjugated with radioisotope are used as another means oftreating human malignancies, particularly hematopoietic malignancies,with a high level of specificity and effectiveness. The most commonlyused isotopes for therapy are the high-energy-emitters, such as ¹³¹I and⁹⁰Y. Recently, ²¹³Bi-labeled anti-CD33 humanized MAb has also beentested in phase I human clinical trials. Reff et al., supra.

[0702] A number of MAbs have been used for therapeutic purposes. Forexample, the use of rituximab (Rituxan™), a recombinant chimericanti-CD20 MAb, for treating certain hematopoietic malignancies wasapproved by the FDA in 1997. Other MAbs that have since been approvedfor therapeutic uses in treating human cancers include: alemtuzumab(Campath-1H™), a humanized rat antibody against CD52; and gemtuzumabozogamicin (Mylotarg™), a calicheamicin-conjugated humanized mouseantCD33 MAb. The FDA is also currently examining the safety and efficacyof several other MAbs for the purpose of site-specific delivery ofcytotoxic agents or radiation, e.g., radiolabeled Zevalin™ and Bexxar™.Reff et al., supra.

[0703] A second important consideration in designing a drug deliverysystem is the accessibility of a target tissue to a therapeutic agent.This is an issue of particular concern in the case of treating a diseaseof the central nervous system (CNS), where the blood-brain barrierprevents the diffusion of macromolecules. Several approaches have beendeveloped to bypass the blood-brain barrier for effective delivery oftherapeutic agents to the CNS.

[0704] The understanding of iron transport mechanism from plasma tobrain provides a useful tool in bypassing the blood-brain barrier (BBB).Iron, transported in plasma by transferrin, is an essential component ofvirtually all types of cells. The brain needs iron for metabolicprocesses and receives iron through transferrin receptors located onbrain capillary endothelial cells via receptor-mediated transcytosis andendocytosis. Moos and Morgan, Cellular and Molecular Neurobiology20:77-95 (2000). Delivery systems based on transferrin-transferrinreceptor interaction have been established for the efficient delivery ofpeptides, proteins, and liposomes into the brain. For example, peptidescan be coupled with a Mab directed against the transferrin receptor toachieve greater uptake by the brain, Moos and Morgan, Supra. Similarly,when coupled with an MAb directed against the transferrin receptor, thetransportation of basic fibroblast growth factor (bFGF) across theblood-brain barrier is enhanced. Song et al., The Journal ofPharmacology and Experimental Therapeutics 301:605-610 (2002); Wu etal., Journal of Drug Targeting 10:239-245 (2002). In addition, aliposomal delivery system for effective transport of the chemotherapydrug, doxorubicin, into C6 glioma has been reported, where transferrinwas attached to the distal ends of liposomal PEG chains. Eavarone etal., J. Biomed. Mater. Res. 51:10-14 (2000). A number of US patents alsorelate to delivery methods bypassing the blood-brain barrier based ontransferrin-transferrin receptor interaction. See e.g., U.S. Pat. Nos.5,154,924; 5,182,107; 5,527,527; 5,833,988; 6,015,555.

[0705] There are other suitable conjugation partners for apharmaceutical agent to bypass the blood-brain barrier. For example,U.S. Pat. Nos. 5,672,683, 5,977,307 and WO 95/02421 relate to a methodof delivering a neuropharmaceutical agent across the blood-brainbarrier, where the agent is administered in the form of a fusion proteinwith a ligand that is reactive with a brain capillary endothelial cellreceptor; WO 99/00150 describes a drug delivery system in which thetransportation of a drug across the blood-brain barrier is facilitatedby conjugation with an MAb directed against human insulin receptor; WO89/10134 describes a chimeric peptide, which includes a peptide capableof crossing the blood brain barrier at a relatively high rate and ahydrophilic neuropeptide incapable of transcytosis, as a means ofintroducing hydrophilic neuropeptides into the brain; WO 01/60411 A1provides a pharmaceutical composition that can easily transport apharmaceutically active ingredient into the brain. The active ingredientis bound to a hibernation-specific protein that is used as a conjugate,and administered with a thyroid hormone or a substance promoting thyroidhormone production. In addition, an alternative route of drug deliveryfor bypassing the blood-brain barrier has been explored. For instance,intranasal delivery of therapeutic agents without the need forconjugation has been shown to be a promising alternative delivery method(Frey, 2002, Drug Delivery Technology, 2(5):46-49).

[0706] In addition to facilitating the transportation of drugs acrossthe blood-brain barrier, transferrin-transferrin receptor interaction isalso useful for specific targeting of certain tumor cells, as many tumorcells overexpress transferrin receptor on their surface. This strategyhas been used for delivering bioactive macromolecules into K562 cellsvia a transferrin conjugate (Wellhoner et al., The Journal of BiologicalChemistry 266:4309-4314 (1991)), and for delivering insulin intoenterocyte-like Caco-2 cells via a transferrin conjugate (Shah and Shen,Journal of Pharmaceutical Sciences 85:1306-1311 (1996)).

[0707] Furthermore, as more becomes known about the functions of variousiron transport proteins, such as lactotransferrin receptor,melanotransferrin, ceruloplasmin, and Divalent Cation Transporter andtheir expression pattern, some of the proteins involved in irontransport mechanism (e.g., melanotransferrin), or their fragments, havebeen found to be similarly effective in assisting therapeutic agentstransport across the blood-brain barrier or targeting specific tissues(WO 02/13843 A2, WO 02/13873 A2). For a review on the use of transferrinand related proteins involved in iron uptake as conjugates in drugdelivery, see Li and Qian, Medical Research Reviews 22:225-250 (2002).

[0708] The concept of tissue-specific delivery of therapeutic agentsgoes beyond the interaction between transferrin and transferrin receptoror their related proteins. For example, a bone-specific delivery systemhas been described in which proteins are conjugated with a bone-seekingaminobisphosphate for improved delivery of proteins to mineralizedtissue. Uludag and Yang, Biotechnol. Prog. 18:604-611 (2002). For areview on this topic, see Vyas et al., Critical Reviews in TherapeuticDrug Carrier System 18:1-76 (2001).

[0709] A variety of linkers may be used in the process of generatingbioconjugates for the purpose of specific delivery of therapeuticagents. Suitable linkers include homo- and heterobifunctionalcross-linking reagents, which may be cleavable by, e.g., acid-catalyzeddissociation, or non-cleavable (see, e.g., Srinivasachar and Neville,Biochemistry 28:2501-2509 (1989); Wellhoner et al., The Journal ofBiological Chemistry 266:4309-4314 (1991)). Interaction between manyknown binding partners, such as biotin and avidin/streptavidin, can alsobe used as a means to join a therapeutic agent and a conjugate partnerthat ensures the specific and effective delivery of the therapeuticagent. Using the methods of the invention, proteins may be used todeliver molecules to intracellular compartments as conjugates. Proteins,peptides, hormones, cytokines, small molecules or the like that bind tospecific cell surface receptors that are internalized after ligandbinding may be used for intracellular targeting of conjugatedtherapeutic compounds. Typically, the receptor-ligand complex isinternalized into intracellular vesicles that are delivered to specificcell compartments, including, but not limited to, the nucleus,mitochondria, golgi, ER, lysosome, and endosome, depending on theintracellular location targeted by the receptor. By conjugating thereceptor ligand with the desired molecule, the drug will be carried withthe receptor-ligand complex and be delivered to the intracellularcompartments normally targeted by the receptor. The drug can thereforebe delivered to a specific intracellular location in the cell where itis needed to treat a disease.

[0710] Many proteins may be used to target therapeutic agents tospecific tissues and organs. Targeting proteins include, but are notlimited to, growth factors (EPO, HGH, EGF, nerve growth factor, FGF,among others), cytokines (GM-CSF, G-CSF, the interferon family,interleukins, among others), hormones (FSH, LH, the steroid families,estrogen, corticosteroids, insulin, among others), serum proteins(albumin, lipoproteins, fetoprotein, human serum proteins, antibodiesand fragments of antibodies, among others), and vitamins (folate,vitamin C, vitamin A, among others). Targeting agents are available thatare specific for receptors on most cells types.

[0711] Contemplated linkage configurations include, but are not limitedto, protein-sugar-linker-sugar-protein and multivalent forms thereof,protein-sugar-linker-protein and multivalent forms thereof,protein-sugar-linker-therapeutic agent, where the therapeutic agentincludes, but are not limited to, small molecules, peptides and lipids.In some embodiments, a hydrolysable linker is used that can behydrolyzed once internalized. An acid labile linker can be used toadvantage where the protein conjugate is internalized into the endosomesor lysosomes which have an acidic pH. Once internalized into theendosome or lysosome, the linker is hydrolyzed and the therapeutic agentis released from the targeting agent.

[0712] In an exemplary embodiment, transferrin is conjugated via alinker to an enzyme or a nucleic acid vector that encoded the enzymedesired to be targeted to a cell that presents transferrin receptors ina patient. The patient could, for example, require enzyme replacementtherapy for that particular enzyme. In particularly preferredembodiments, the enzyme is one that is lacking in a patient with alysosomal storage disease (see Table 5). Once in circulation, thetransferrin-enzyme conjugate is linked to transferrin receptors and isinternalized in early endosomes (Xing et al., 1998, Biochem. J. 336:667;Li et al., 2002, Trends in Pharmcol. Sci. 23:206; Suhaila et al., 1998,J. Biol. Chem. 273:14355). Other contemplated targeting agents that arerelated to transferrin include, but are not limited to, lactotransferrin(lactoferrin), melanotransferrin (p97), ceruloplasmin, and divalentcation transporter.

[0713] In another exemplary embodiment, transferrin-dystrophinconjugates would enter endosomes by the transferrin pathway. Once there,the dystrophin is released due to a hydrolysable linker which can thenbe taken to the intracellular compartment where it is required. Thisembodiment may be used to treat a patient with muscular dystrophy bysupplementing a genetically defective dystrophin gene and/or proteinwith the functional dystrophin peptide connected to the transferrin.

[0714] E. Therapeutic Moieties

[0715] In another preferred embodiment, the modified sugar includes atherapeutic moiety. Those of skill in the art will appreciate that thereis overlap between the category of therapeutic moieties andbiomolecules; many biomolecules have therapeutic properties orpotential.

[0716] The therapeutic moieties can be agents already accepted forclinical use or they can be drugs whose use is experimental, or whoseactivity or mechanism of action is under investigation. The therapeuticmoieties can have a proven action in a given disease state or can beonly hypothesized to show desirable action in a given disease state. Ina preferred embodiment, the therapeutic moieties are compounds, whichare being screened for their ability to interact with a tissue ofchoice. Therapeutic moieties, which are useful in practicing the instantinvention include drugs from a broad range of drug classes having avariety of pharmacological activities. In some embodiments, it ispreferred to use therapeutic moieties that are not sugars. An exceptionto this preference is the use of a sugar that is modified by covalentattachment of another entity, such as a PEG, biomolecule, therapeuticmoiety, diagnostic moiety and the like. In an exemplary embodiment, anantisense nucleic acid moeity is conjugated to a linker arm which isattached to the targeting moiety. In another exemplary embodiment, atherapeutic sugar moiety is conjugated to a linker arm and thesugar-linker arm cassette is subsequently conjugated to a peptide via amethod of the invention.

[0717] Methods of conjugating therapeutic and diagnostic agents tovarious other species are well known to those of skill in the art. See,for example Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, SanDiego, 1996; and Dunn et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERYSYSTEMS, ACS Symposium Series Vol. 469, American Chemical Society,Washington, D.C. 1991.

[0718] In an exemplary embodiment, the therapeutic moiety is attached tothe modified sugar via a linkage that is cleaved under selectedconditions. Exemplary conditions include, but are not limited to, aselected pH (e.g., stomach, intestine, endocytotic vacuole), thepresence of an active enzyme (e.g., esterase, protease, reductase,oxidase), light, heat and the like. Many cleavable groups are known inthe art. See, for example, Jung et al., Biochem. Biophys. Acta, 761:152-162 (1983); Joshi et al., J. Biol. Chem., 265: 14518-14525 (1990);Zarling et al., J. Immunol., 124: 913-920 (1980); Bouizar et al., Eur.J. Biochem., 155: 141-147 (1986); Park et al., J. Biol. Chem., 261:205-210 (1986); Browning et al., J. Immunol., 143: 1859-1867 (1989).

[0719] Classes of useful therapeutic moieties include, for example,non-steroidal anti-inflammatory drugs (NSAIDS). The NSAIDS can, forexample, be selected from the following categories: (e.g., propionicacid derivatives, acetic acid derivatives, fenamic acid derivatives,biphenylcarboxylic acid derivatives and oxicams); steroidalanti-inflammatory drugs including hydrocortisone and the like;adjuvants; antihistaminic drugs (e.g., chlorpheniramine, triprolidine);antitussive drugs (e.g., dextromethorphan, codeine, caramiphen andcarbetapentane); antipruritic drugs (e.g., methdilazine andtrimeprazine); anticholinergic drugs (e.g., scopolamine, atropine,homatropine, levodopa); anti-emetic and antinauseant drugs (e.g.,cyclizine, meclizine, chlorpromazine, buclizine); anorexic drugs (e.g.,benzphetamine, phentermine, chlorphentermine, fenfluramine); centralstimulant drugs (e.g., amphetamine, methamphetamine, dextroamphetamineand methylphenidate); antiarrhythmic drugs (e.g., propanolol,procainamide, disopyramide, quinidine, encainide); β-adrenergic blockerdrugs (e.g., metoprolol, acebutolol, betaxolol, labetalol and timolol);cardiotonic drugs (e.g., milrinone, amrinone and dobutamine);antihypertensive drugs (e.g., enalapril, clonidine, hydralazine,minoxidil, guanadrel, guanethidine); diuretic drugs (e.g., amiloride andhydrochlorothiazide); vasodilator drugs (e.g., diltiazem, amiodarone,isoxsuprine, nylidrin, tolazoline and verapamil); vasoconstrictor drugs(e.g., dihydroergotamine, ergotamine and methylsergide); antiulcer drugs(e.g., ranitidine and cimetidine); anesthetic drugs (e.g., lidocaine,bupivacaine, chloroprocaine, dibucaine); antidepressant drugs (e.g.,imipramine, desipramine, amitryptiline, nortryptiline); tranquilizer andsedative drugs (e.g., chlordiazepoxide, benacytyzine, benzquinamide,flurazepam, hydroxyzine, loxapine and promazine); antipsychotic drugs(e.g., chlorprothixene, fluphenazine, haloperidol, molindone,thioridazine and trifluoperazine); antimicrobial drugs (antibacterial,antifungal, antiprotozoal and antiviral drugs).

[0720] Classes of useful therapeutic moieties include adjuvants. Theadjuvants can, for example, be selected from keyhole lymphet hemocyaninconjugates, monophosphoryl lipid A, mycoplasma-derived lipopeptideMALP-2, cholera toxin B subunit, Escherichia coli heat-labile toxin,universal T helper epitope from tetanus toxoid, interleukin-12, CpGoligodeoxynucleotides, dimethyldioctadecylammonium bromide,cyclodextrin, squalene, aluminum salts, meningococcal outer membranevesicle (OMV), montanide ISA, TiterMax™ (available from Sigma, St. LouisMo.), nitrocellulose absorption, immune-stimulating complexes such asQuil A, Gerbu™ adjuvant (Gerbu Biotechnik, Kirchwald, Germany), threonylmuramyl dipeptide, thymosin alpha, bupivacaine, GM-CSF, IncompleteFreund's Adjuvant, MTP-PE/MF59 (Ciba/Geigy, Basel, Switzerland),polyphosphazene, saponin derived from the soapbark tree Quillajasaponaria, and Syntex adjuvant formulation (Biocine, Emeryville,Calif.), among others well known to those in the art.

[0721] Antimicrobial drugs which are preferred for incorporation intothe present composition include, for example, pharmaceuticallyacceptable salts of β-lactam drugs, quinolone drugs, ciprofloxacin,norfloxacin, tetracycline, erythromycin, amikacin, triclosan,doxycycline, capreomycin, chlorhexidine, chlortetracycline,oxytetracycline, clindamycin, ethambutol, hexamidine isothionate,metronidazole, pentamidine, gentamycin, kanamycin, lineomycin,methacycline, methenamine, minocycline, neomycin, netilmycin,paromomycin, streptomycin, tobramycin, miconazole and amantadine.

[0722] Other drug moieties of use in practicing the present inventioninclude antineoplastic drugs (e.g., antiandrogens (e.g., leuprolide orflutamide), cytocidal agents (e.g., adriamycin, doxorubicin, taxol,cyclophosphamide, busulfan, cisplatin, α-2-interferon) anti-estrogens(e.g., tamoxifen), antimetabolites (e.g., fluorouracil, methotrexate,mercaptopurine, thioguanine). Also included within this class areradioisotope-based agents for both diagnosis and therapy, and conjugatedtoxins, such as ricin, geldanamycin, mytansin, CC-1065, C-1027, theduocarmycins, calicheamycin and related structures and analoguesthereof, and the toxins listed in Table 2.

[0723] The therapeutic moiety can also be a hormone (e.g.,medroxyprogesterone, estradiol, leuprolide, megestrol, octreotide orsomatostatin); muscle relaxant drugs (e.g., cinnamedrine,cyclobenzaprine, flavoxate, orphenadrine, papaverine, mebeverine,idaverine, ritodrine, diphenoxylate, dantrolene and azumolen);antispasmodic drugs; bone-active drugs (e.g., diphosphonate andphosphonoalkylphosphinate drug compounds); endocrine modulating drugs(e.g., contraceptives (e.g., ethinodiol, ethinyl estradiol,norethindrone, mestranol, desogestrel, medroxyprogesterone), modulatorsof diabetes (e.g., glyburide or chlorpropamide), anabolics, such astestolactone or stanozolol, androgens (e.g., methyltestosterone,testosterone or fluoxymesterone), antidiuretics (e.g., desmopressin) andcalcitonins).

[0724] Also of use in the present invention are estrogens (e.g.,diethylstilbesterol), glucocorticoids (e.g., triamcinolone,betamethasone, etc.) and progesterones, such as norethindrone,ethynodiol, norethindrone, levonorgestrel; thyroid agents (e.g.,liothyronine or levothyroxine) or anti-thyroid agents (e.g.,methimazole); antihyperprolactinemic drugs (e.g., cabergoline); hormonesuppressors (e.g., danazol or goserelin), oxytocics (e.g.,methylergonovine or oxytocin) and prostaglandins, such as mioprostol,alprostadil or dinoprostone, can also be employed.

[0725] Other useful modifying groups include immunomodulating drugs(e.g., antihistamines, mast cell stabilizers, such as Iodoxamide and/orcromolyn, steroids (e.g., triamcinolone, beclomethazone, cortisone,dexamethasone, prednisolone, methylprednisolone, beclomethasone, orclobetasol), histamine H2 antagonists (e.g., famotidine, cimetidine,ranitidine), immunosuppressants (e.g., azathioprine, cyclosporin), etc.Groups with anti-inflammatory activity, such as sulindac, etodolac,ketoprofen and ketorolac, are also of use. Other drugs of use inconjunction with the present invention will be apparent to those ofskill in the art.

[0726] Classes of useful therapeutic moieties include, for example,antisense drugs and also naked DNA. The antisense drugs can be selectedfrom for example Affinitak (ISIS, Carlsbad, Calif.) and Genasense™ (fromGenta, Berkeley Heights, N.J.). Naked DNA can be delivered as a genetherapy therapeutic for example with the DNA encoding for examplefactors VIII and IX for treatment of hemophilia disorders.

[0727] F. Preparation of Modified Sugars

[0728] Modified sugars useful in forming the conjugates of the inventionare discussed herein. The discussion focuses on preparing a sugarmodified with a water-soluble polymer for clarity of illustration. Inparticular, the discussion focuses on the preparation of modified sugarsthat include a poly(ethylene glycol) moiety. Those of skill willappreciate that the methods set forth herein are broadly applicable tothe preparation of modified sugars, therefore, the discussion should notbe interpreted as limiting the scope of the invention.

[0729] In general, the sugar moiety and the modifying group are linkedtogether through the use of reactive groups, which are typicallytransformed by the linking process into a new organic functional groupor unreactive species. The sugar reactive functional group(s), islocated at any position on the sugar moiety. Reactive groups and classesof reactions useful in practicing the present invention are generallythose that are well known in the art of bioconjugate chemistry.Currently favored classes of reactions available with reactive sugarmoieties are those, which proceed under relatively mild conditions.These include, but are not limited to nucleophilic substitutions (e.g.,reactions of amines and alcohols with acyl halides, active esters),electrophilic substitutions (e.g., enamine reactions) and additions tocarbon-carbon and carbon-heteroatom multiple bonds (e.g., Michaelreaction, Diels-Alder addition). These and other useful reactions arediscussed in, for example, Smith and March, ADVANCED ORGANIC CHEMISTRY,5th Ed., John Wiley & Sons, New York, 2001; Hermanson, BIOCONJUGATETECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al.,MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198,American Chemical Society, Washington, D.C., 1982.

[0730] Useful reactive functional groups pendent from a sugar nucleus ormodifying group include, but are not limited to:

[0731] (a) carboxyl groups and various derivatives thereof including,but not limited to, N-hydroxysuccinimide esters, N-hydroxybenzotriazoleesters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters,alkyl, alkenyl, alkynyl and aromatic esters;

[0732] (b) hydroxyl groups, which can be converted to, e.g., esters,ethers, aldehydes, etc.

[0733] (c) haloalkyl groups, wherein the halide can be later displacedwith a nucleophilic group such as, for example, an amine, a carboxylateanion, thiol anion, carbanion, or an alkoxide ion, thereby resulting inthe covalent attachment of a new group at the functional group of thehalogen atom;

[0734] (d) dienophile groups, which are capable of participating inDiels-Alder reactions such as, for example, maleimido groups;

[0735] (e) aldehyde or ketone groups, such that subsequentderivatization is possible via formation of carbonyl derivatives suchas, for example, imines, hydrazones, semicarbazones or oximes, or viasuch mechanisms as Grignard addition or alkyllithium addition;

[0736] (f) sulfonyl halide groups for subsequent reaction with amines,for example, to form sulfonamides;

[0737] (g) thiol groups, which can be, for example, converted todisulfides or reacted with alkyl and acyl halides;

[0738] (h) amine or sulfhydryl groups, which can be, for example,acylated, alkylated or oxidized;

[0739] (i) alkenes, which can undergo, for example, cycloadditions,acylation, Michael addition, etc; and

[0740] (j) epoxides, which can react with, for example, amines andhydroxyl compounds.

[0741] The reactive functional groups can be chosen such that they donot participate in, or interfere with, the reactions necessary toassemble the reactive sugar nucleus or modifying group. Alternatively, areactive functional group can be protected from participating in thereaction by the presence of a protecting group. Those of skill in theart understand how to protect a particular functional group such that itdoes not interfere with a chosen set of reaction conditions. Forexamples of useful protecting groups, see, for example, Greene et al.,PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York,1991.

[0742] In the discussion that follows, a number of specific examples ofmodified sugars that are useful in practicing the present invention areset forth. In the exemplary embodiments, a sialic acid derivative isutilized as the sugar nucleus to which the modifying group is attached.The focus of the discussion on sialic acid derivatives is for clarity ofillustration only and should not be construed to limit the scope of theinvention. Those of skill in the art will appreciate that a variety ofother sugar moieties can be activated and derivatized in a manneranalogous to that set forth using sialic acid as an example. Forexample, numerous methods are available for modifying galactose,glucose, N-acetylgalactosamine and fucose to name a few sugarsubstrates, which are readily modified by art recognized methods. See,for example, Elhalabi et al., Curr. Med. Chem. 6: 93 (1999); and Schaferet al., J. Org. Chem. 65: 24 (2000).

[0743] In an exemplary embodiment, the peptide that is modified by amethod of the invention is a peptide that is produced in mammalian cells(e.g., CHO cells) or in a transgenic animal and thus, contains N- and/orO-linked oligosaccharide chains, which are incompletely sialylated. Theoligosaccharide chains of the glycopeptide lacking a sialic acid andcontaining a terminal galactose residue can be PEGylated, PPGylated orotherwise modified with a modified sialic acid.

[0744] In Scheme 4, the mannosamine glycoside 1, is treated with theactive ester of a protected amino acid (e.g., glycine) derivative,converting the sugar amine residue into the corresponding protectedamino acid amide adduct. The adduct is treated with an aldolase to formthe sialic acid 2. Compound 2 is converted to the corresponding CMPderivative by the action of CMP-SA synthetase, followed by catalytichydrogenation of the CMP derivative to produce compound 3. The amineintroduced via formation of the glycine adduct is utilized as a locus ofPEG or PPG attachment by reacting compound 3 with an activated PEG orPPG derivative (e.g., PEG-C(O)NHS, PPG-C(O)NHS), producing 4 or 5,respectively.

[0745] Table 3 sets forth representative examples of sugarmonophosphates that are derivatized with a PEG or PPG moiety. Certain ofthe compounds of Table 3 are prepared by the method of Scheme 1. Otherderivatives are prepared by art-recognized methods. See, for example,Keppler et al., Glycobiology 11: 11R (2001); and Charter et al.,Glycobiology 10: 1049 (2000)). Other amine reactive PEG and PPGanalogues are commercially available, or they can be prepared by methodsreadily accessible to those of skill in the art. TABLE 3 Examples ofsugar monophosphates that are derivatized with a PEG or PPG moiety

CMP-KDN-5-O-R

CMP-NeuAc-9-NH-R

CMP-NeuAc-8-O-R

CMP-NeuAc-8-NH-R

CMP-NeuAc-7-O-R

CMP-NeuAc-7-NH-R

CMP-NeuAc-4-O-R

CMP-NeuAc-4-NH-R

CMP-SA-5-NH-R

CMP-NeuAc-9-O-R

[0746] The modified sugar phosphates of use in practicing the presentinvention can be substituted in other positions as well as those setforth above. “i” may be Na or another salt and “i” may beinterchangeable with Na. Presently preferred substitutions of sialicacid are set forth in Formula 5.

[0747] in which X is a linking group, which is preferably selected from—O—, —N(H)—, —S, CH₂—, and N(R)₂, in which each R is a memberindependently selected from R¹-R⁵. “i” may be Na or another salt, and Namay be interchangeable with “i: The symbols Y, Z, A and B each representa group that is selected from the group set forth above for the identityof X. X, Y, Z, A and B are each independently selected and, therefore,they can be the same or different. The symbols R¹, R², R³, R⁴ and R⁵represent H, polymers, a water-soluble polymer, therapeutic moiety,biomolecule or other moiety. The symbol R6 represents H, OH, or apolymer. Alternatively, these symbols represent a linker that is linkedto a polymer, water-soluble polymer, therapeutic moiety, biomolecule orother moiety.

[0748] In another exemplary embodiment, a mannosamine is simultaneouslyacylated and activated for a nucleophilic substitution by the use ofchloroacetic anhydride as set forth in Scheme 5. In each of the schemespresented in this section, i⁺ or Na⁺ can be interchangeable, wherein thesalt can be sodium, or can be any other suitable salt.

[0749] The resulting chloro-derivatized glycan is contacted withpyruvate in the presence of an aldolase, forming a chloro-derivatizedsialic acid. The corresponding nucleotide sugar is prepared by contactedthe sialic acid derivative with an appropriate nucleotide triphosphatesand a synthetase. The chloro group on the sialic acid moiety is thendisplaced with a nucleophilic PEG derivative, such as thio-PEG.

[0750] In a further exemplary embodiment, as shown is Scheme 6, amannosamine is acylated with a bis-HOBT dicarboxylate, producing thecorresponding amido-alkyl-carboxylic acid, which is subsequentlyconverted to a sialic acid derivative. The sialic acid derivative isconverted to a nucleotide sugar, and the carboxylic acid is activatedand reacted with a nucleophilic PEG derivative, such as amino-PEG.

[0751] In another exemplary embodiment, set forth in Scheme 7, amine-and carboxyl-protected neuraminic acid is activated by converting theprimary hydroxyl group to the corresponding p-toluenesulfonate ester,and the methyl ester is cleaved. The activated neuraminic acid isconverted to the corresponding nucleotide sugar, and the activatinggroup is displaced by a nucleophilic PEG species, such as thio-PEG.

[0752] In yet a further exemplary embodiment, as set forth in Scheme 8,the primary hydroxyl moiety of an amine- and carboxyl-protectedneuraminic acid derivative is alkylated using an electrophilic PEG, suchas chloro-PEG. The methyl ester is subsequently cleaved and thePEG-sugar is converted to a nucleotide sugar.

[0753] Glycans other than sialic acid can be derivatized with PEG usingthe methods set forth herein. The derivatized glycans, themselves, arealso within the scope of the invention. Thus, Scheme 9 provides anexemplary synthetic route to a PEGylated galactose nucleotide sugar. Theprimary hydroxyl group of galactose is activated as the correspondingtoluenesulfonate ester, which is subsequently converted to a nucleotidesugar.

[0754] Scheme 10 sets forth an exemplary route for preparing agalactose-PEG derivative that is based upon a galactose-6-amine moiety.Thus, galactosamine is converted to a nucleotide sugar, and the aminemoiety of galactosamine is functionalized with an active PEG derivative.

[0755] Scheme 11 provides another exemplary route to galactosederivatives. The starting point for Scheme 11 is galactose-2-amine,which is converted to a nucleotide sugar. The amine moiety of thenucleotide sugar is the locus for attaching a PEG derivative, such asMethoxy-PEG (mPEG) carboxylic acid.

[0756] Exemplary moieties attached to the conjugates disclosed hereininclude, but are not limited to, PEG derivatives (e.g., acyl-PEG,acyl-alkyl-PEG, alkyl-acyl-PEG carbamoyl-PEG, aryl-PEG, alkyl-PEG), PPGderivatives (e.g., acyl-PPG, acyl-alkyl-PPG, alkyl-acyl-PPGcarbamoyl-PPG, aryl-PPG), polyapartic acid, polyglutamate, polylysine,therapeutic moieties, diagnostic moieties, mannose-6-phosphate, heparin,heparan, SLex, mannose, mannose-6-phosphate, Sialyl Lewis X, FGF, VFGF,proteins (e.g., transferrin), chondroitin, keratan, dermatan, dextran,modified dextran, amylose, bisphosphate, poly-SA, hyaluronic acid,keritan, albumin, integrins, antennary oligosaccharides, peptides andthe like. Methods of conjugating the various modifying groups to asaccharide moiety are readily accessible to those of skill in the art(POLY (ETHYLENE GLYCOL CHEMISTRY: BIOTECHNICAL AND BIOMEDICALAPPLICATIONS, J. Milton Harris, Ed., Plenum Pub. Corp., 1992; POLY(ETHYLENE GLYCOL) CHEMICAL AND BIOLOGICAL APPLICATIONS, J. MiltonHarris, Ed., ACS Symposium Series No. 680, American Chemical Society,1997; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego,1996; and Dunn et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS,ACS Symposium Series Vol. 469, American Chemical Society, Washington,D.C. 1991).

[0757] Purification of Sugars, Nucleotide Sugars and Derivatives

[0758] The nucleotide sugars and derivatives produced by the aboveprocesses can be used without purification. However, it is usuallypreferred to recover the product. Standard, well-known techniques forrecovery of glycosylated saccharides such as thin or thick layerchromatography, column chromatography, ion exchange chromatography, ormembrane filtration can be used. It is preferred to use membranefiltration, more preferably utilizing a reverse osmotic membrane, or oneor more column chromatographic techniques for the recovery as isdiscussed hereinafter and in the literature cited herein. For instance,membrane filtration wherein the membranes have molecular weight cutoffof about 3000 to about 10,000 can be used to remove proteins forreagents having a molecular weight of less than 10,000 Da. Membranefiltration or reverse osmosis can then be used to remove salts and/orpurify the product saccharides (see, e.g., WO 98/15581). Nanofiltermembranes are a class of reverse osmosis membranes that pass monovalentsalts but retain polyvalent salts and uncharged solutes larger thanabout 100 to about 2,000 Daltons, depending upon the membrane used.Thus, in a typical application, saccharides prepared by the methods ofthe present invention will be retained in the membrane and contaminatingsalts will pass through.

[0759] G. Cross-Linking Groups

[0760] Preparation of the modified sugar for use in the methods of thepresent invention includes attachment of a modifying group to a sugarresidue and forming a stable adduct, which is a substrate for aglycosyltransferase. Thus, it is often preferred to use a cross-linkingagent to conjugate the modifying group and the sugar. Exemplarybifunctional compounds which can be used for attaching modifying groupsto carbohydrate moieties include, but are not limited to, bifunctionalpoly(ethylene glycols), polyamides, polyethers, polyesters and the like.General approaches for linking carbohydrates to other molecules areknown in the literature. See, for example, Lee et al., Biochemistry 28:1856 (1989); Bhatia et al., Anal. Biochem. 178: 408 (1989); Janda etal., J. Am. Chem. Soc. 112: 8886 (1990) and Bednarski et al., WO92/18135. In the discussion that follows, the reactive groups aretreated as benign on the sugar moiety of the nascent modified sugar. Thefocus of the discussion is for clarity of illustration. Those of skillin the art will appreciate that the discussion is relevant to reactivegroups on the modifying group as well.

[0761] An exemplary strategy involves incorporation of a protectedsulfhydryl onto the sugar using the heterobifunctional crosslinker SPDP(n-succinimidyl-3-(2-pyridyldithio)propionate and then deprotecting thesulfhydryl for formation of a disulfide bond with another sulfhydryl onthe modifying group.

[0762] If SPDP detrimentally affects the ability of the modified sugarto act as a glycosyltransferase substrate, one of an array of othercrosslinkers such as 2-iminothiolane or N-succinimidylS-acetylthioacetate (SATA) is used to form a disulfide bond.2-iminothiolane reacts with primary amines, instantly incorporating anunprotected sulfhydryl onto the amine-containing molecule. SATA alsoreacts with primary amines, but incorporates a protected sulfhydryl,which is later deacetylated using hydroxylamine to produce a freesulfhydryl. In each case, the incorporated sulfhydryl is free to reactwith other sulfhydryls or protected sulfhydryl, like SPDP, forming therequired disulfide bond.

[0763] The above-described strategy is exemplary, and not limiting, oflinkers of use in the invention. Other crosslinkers are available thatcan be used in different strategies for crosslinking the modifying groupto the peptide. For example, TPCH(S-(2-thiopyridyl)-L-cysteine hydrazideand TPMPH ((S-(2-thiopyridyl) mercapto-propionohydrazide) react withcarbohydrate moieties that have been previously oxidized by mildperiodate treatment, thus forming a hydrazone bond between the hydrazideportion of the crosslinker and the periodate generated aldehydes. TPCHand TPMPH introduce a 2-pyridylthione protected sulfhydryl group ontothe sugar, which can be deprotected with DTT and then subsequently usedfor conjugation, such as forming disulfide bonds between components.

[0764] If disulfide bonding is found unsuitable for producing stablemodified sugars, other crosslinkers may be used that incorporate morestable bonds between components. The heterobifunctional crosslinkersGMBS (N-gama-malimidobutyryloxy)succinimide) and SMCC (succinimidyl4-(N-maleimido-methyl)cyclohexane) react with primary amines, thusintroducing a maleimide group onto the component. The maleimide groupcan subsequently react with sulfhydryls on the other component, whichcan be introduced by previously mentioned crosslinkers, thus forming astable thioether bond between the components. If steric hindrancebetween components interferes with either component's activity or theability of the modified sugar to act as a glycosyltransferase substrate,crosslinkers can be used which introduce long spacer arms betweencomponents and include derivatives of some of the previously mentionedcrosslinkers (i.e., SPDP). Thus, there is an abundance of suitablecrosslinkers, which are useful; each of which is selected depending onthe effects it has on optimal peptide conjugate and modified sugarproduction.

[0765] A variety of, reagents are used to modify the components of themodified sugar with intramolecular chemical crosslinks (for reviews ofcrosslinking reagents and crosslinking procedures see: Wold, F., Meth.Enzymol. 25: 623-651, 1972; Weetall, H. H., and Cooney, D. A., In:ENZYMES AS DRUGS. (Holcenberg, and Roberts, eds.) pp. 395-442, Wiley,New York, 1981; Ji, T. H., Meth. Enzymol. 91: 580-609, 1983; Mattson etal., Mol. Biol. Rep. 17: 167-183, 1993, all of which are incorporatedherein by reference). Preferred crosslinking reagents are derived fromvarious zero-length, homo-bifunctional, and hetero-bifunctionalcrosslinking reagents. Zero-length crosslinking reagents include directconjugation of two intrinsic chemical groups with no introduction ofextrinsic material. Agents that catalyze formation of a disulfide bondbelong to this category. Another example is reagents that inducecondensation of a carboxyl and a primary amino group to form an amidebond such as carbodiimides, ethylchloroformate, Woodward's reagent K(2-ethyl-5-phenylisoxazolium-3′-sulfonate), and carbonyldiimidazole. Inaddition to these chemical reagents, the enzyme transglutaminase(glutamyl-peptide γ-glutamyltransferase; EC 2.3.2.13) may be used aszero-length crosslinking reagent. This enzyme catalyzes acyl transferreactions at carboxamide groups of protein-linked glutaminyl residues,usually with a primary amino group as substrate. Preferred homo- andhetero-bifunctional reagents contain two identical or two dissimilarsites, respectively, which may be reactive for amino, sulfhydryl,guanidino, indole, or nonspecific groups.

[0766] 2. Preferred Specific Sites in Crosslinking Reagents

[0767] a. Amino-Reactive Groups

[0768] In one preferred embodiment, the sites on the cross-linker areamino-reactive groups. Useful non-limiting examples of amino-reactivegroups include N-hydroxysuccinimide (NHS) esters, imidoesters,isocyanates, acylhalides, arylazides, p-nitrophenyl esters, aldehydes,and sulfonyl chlorides.

[0769] NHS esters react preferentially with the primary (includingaromatic) amino groups of a modified sugar component. The imidazolegroups of histidines are known to compete with primary amines forreaction, but the reaction products are unstable and readily hydrolyzed.The reaction involves the nucleophilic attack of an amine on the acidcarboxyl of an NHS ester to form an amide, releasing theN-hydroxysuccinimide. Thus, the positive charge of the original aminogroup is lost.

[0770] Imidoesters are the most specific acylating reagents for reactionwith the amine groups of the modified sugar components. At a pH between7 and 10, imidoesters react only with primary amines. Primary aminesattack imidates nucleophilically to produce an intermediate that breaksdown to amidine at high pH or to a new imidate at low pH. The newimidate can react with another primary amine, thus crosslinking twoamino groups, a case of a putatively monofunctional imidate reactingbifunctionally. The principal product of reaction with primary amines isan amidine that is a stronger base than the original amine. The positivecharge of the original amino group is therefore retained.

[0771] Isocyanates (and isothiocyanates) react with the primary aminesof the modified sugar components to form stable bonds. Their reactionswith sulfhydryl, imidazole, and tyrosyl groups give relatively unstableproducts.

[0772] Acylazides are also used as amino-specific reagents in whichnucleophilic amines of the affinity component attack acidic carboxylgroups under slightly alkaline conditions, e.g. pH 8.5.

[0773] Arylhalides such as 1,5-difluoro-2,4-dinitrobenzene reactpreferentially with the amino groups and tyrosine phenolic groups ofmodified sugar components, but also with sulfhydryl and imidazolegroups.

[0774] p-Nitrophenyl esters of mono- and dicarboxylic acids are alsouseful amino-reactive groups. Although the reagent specificity is notvery high, α- and ε-amino groups appear to react most rapidly.

[0775] Aldehydes such as glutaraldehyde react with primary amines ofmodified sugar. Although unstable Schiff bases are formed upon reactionof the amino groups with the aldehydes of the aldehydes, glutaraldehydeis capable of modifying the modified sugar with stable crosslinks. At pH6-8, the pH of typical crosslinking conditions, the cyclic polymersundergo a dehydration to form α-β unsaturated aldehyde polymers. Schiffbases, however, are stable, when conjugated to another double bond. Theresonant interaction of both double bonds prevents hydrolysis of theSchiff linkage. Furthermore, amines at high local concentrations canattack the ethylenic double bond to form a stable Michael additionproduct.

[0776] Aromatic sulfonyl chlorides react with a variety of sites of themodified sugar components, but reaction with the amino groups is themost important, resulting in a stable sulfonamide linkage.

[0777] b. Sulfhydryl-Reactive Groups

[0778] In another preferred embodiment, the sites aresulfhydryl-reactive groups. Useful, non-limiting examples ofsulfhydryl-reactive groups include maleimides, alkyl halides, pyridyldisulfides, and thiophthalimides.

[0779] Maleimides react preferentially with the sulfhydryl group of themodified sugar components to form stable thioether bonds. They alsoreact at a much slower rate with primary amino groups and the imidazolegroups of histidines. However, at pH 7 the maleimide group can beconsidered a sulfhydryl-specific group, since at this pH the reactionrate of simple thiols is 1000-fold greater than that of thecorresponding amine.

[0780] Alkyl halides react with sulfhydryl groups, sulfides, imidazoles,and amino groups. At neutral to slightly alkaline pH, however, alkylhalides react primarily with sulfhydryl groups to form stable thioetherbonds. At higher pH, reaction with amino groups is favored.

[0781] Pyridyl disulfides react with free sulfhydryls via disulfideexchange to give mixed disulfides. As a result, pyridyl disulfides arethe most specific sulfhydryl-reactive groups.

[0782] Thiophthalimides react with free sulfhydryl groups to formdisulfides.

[0783] c. Carboxyl-Reactive Residue

[0784] In another embodiment, carbodiimides soluble in both water andorganic solvent, are used as carboxyl-reactive reagents. These compoundsreact with free carboxyl groups forming a pseudourea that can thencoupled to available amines yielding an amide linkage. Procedures tomodify a carboxyl group with carbodiimide is well know in the art (see,Yamada et al., Biochemistry 20: 4836-4842, 1981).

[0785] 3. Preferred Nonspecific Sites in Crosslinking Reagents

[0786] In addition to the use of site-specific reactive moieties, thepresent invention contemplates the use of non-specific reactive groupsto link the sugar to the modifying group.

[0787] Exemplary non-specific cross-linkers include photoactivatablegroups, completely inert in the dark, which are converted to reactivespecies upon absorption of a photon of appropriate energy. In onepreferred embodiment, photoactivatable groups are selected fromprecursors of nitrenes generated upon heating or photolysis of azides.Electron-deficient nitrenes are extremely reactive and can react with avariety of chemical bonds including N—H, O—H, C—H, and C═C. Althoughthree types of azides (aryl, alkyl, and acyl derivatives) may beemployed, arylazides are presently preferred. The reactivity ofarylazides upon photolysis is better with N—H and O—H than C—H bonds.Electron-deficient arylnitrenes rapidly ring-expand to formdehydroazepines, which tend to react with nucleophiles, rather than formC—H insertion products. The reactivity of arylazides can be increased bythe presence of electron-withdrawing substituents such as nitro orhydroxyl groups in the ring. Such substituents push the absorptionmaximum of arylazides to longer wavelength. Unsubstituted arylazideshave an absorption maximum in the range of 260-280 nm, while hydroxy andnitroarylazides absorb significant light beyond 305 nm. Therefore,hydroxy and nitroarylazides are most preferable since they allow toemploy less harmful photolysis conditions for the affinity componentthan unsubstituted arylazides.

[0788] In another preferred embodiment, photoactivatable groups areselected from fluorinated arylazides. The photolysis products offluorinated arylazides are arylnitrenes, all of which undergo thecharacteristic reactions of this group, including C—H bond insertion,with high efficiency (Keana et al., J. Org. Chem. 55: 3640-3647, 1990).

[0789] In another embodiment, photoactivatable groups are selected frombenzophenone residues. Benzophenone reagents generally give highercrosslinking yields than arylazide reagents.

[0790] In another embodiment, photoactivatable groups are selected fromdiazo compounds, which form an electron-deficient carbene uponphotolysis. These carbenes undergo a variety of reactions includinginsertion into C—H bonds, addition to double bonds (including aromaticsystems), hydrogen attraction and coordination to nucleophilic centersto give carbon ions.

[0791] In still another embodiment, photoactivatable groups are selectedfrom diazopyruvates. For example, the p-nitrophenyl ester ofp-nitrophenyl diazopyruvate reacts with aliphatic amines to givediazopyruvic acid amides that undergo ultraviolet photolysis to formaldehydes. The photolyzed diazopyruvate-modified affinity component willreact like formaldehyde or glutaraldehyde forming crosslinks.

[0792] 4. Homobifunctional Reagents

[0793] a. Homobifunctional Crosslinkers Reactive With Primary Amines

[0794] Synthesis, properties, and applications of amine-reactivecross-linkers are commercially described in the literature (for reviewsof crosslinking procedures and reagents, see above). Many reagents areavailable (e.g., Pierce Chemical Company, Rockford, Ill.; Sigma ChemicalCompany, St. Louis, Mo.; Molecular Probes, Inc., Eugene, Oreg.).

[0795] Preferred, non-limiting examples of homobifunctional NHS estersinclude disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS),bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartarate (DST),disulfosuccinimidyl tartarate (sulfo-DST),bis-2-(succinimidooxycarbonyloxy)ethylsulfone (BSOCOES),bis-2-(sulfosuccinimidooxy-carbonyloxy)ethylsulfone (sulfo-BSOCOES),ethylene glycolbis(succinimidylsuccinate) (EGS), ethyleneglycolbis(sulfosuccinimidylsuccinate) (sulfo-EGS),dithiobis(succinimidyl-propionate (DSP), anddithiobis(sulfosuccinimidylpropionate (sulfo-DSP). Preferred,non-limiting examples of homobifunctional imidoesters include dimethylmalonimidate (DMM), dimethyl succinimidate (DMSC), dimethyl adipimidate(DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS),dimethyl-3,3′-oxydipropionimidate (DODP),dimethyl-3,3′-(methylenedioxy)dipropionimidate (DMDP),dimethyl-,3′-(dimethylenedioxy)dipropionimidate (DDDP),dimethyl-3,3′-(tetramethylenedioxy)-dipropionimidate (DTDP), anddimethyl-3,3′-dithiobispropionimidate (DTBP).

[0796] Preferred, non-limiting examples of homobifunctionalisothiocyanates include: p-phenylenediisothiocyanate (DITC), and4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS).

[0797] Preferred, non-limiting examples of homobifunctional isocyanatesinclude xylene-diisocyanate, toluene-2,4-diisocyanate,toluene-2-isocyanate-4-isothiocyanate,3-methoxydiphenylmethane-4,4′-diisocyanate,2,2′-dicarboxy-4,4′-azophenyldiisocyanate, andhexamethylenediisocyanate.

[0798] Preferred, non-limiting examples of homobifunctional arylhalidesinclude 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and4,4′-difluoro-3,3′-dinitrophenyl-sulfone.

[0799] Preferred, non-limiting examples of homobifunctional aliphaticaldehyde reagents include glyoxal, malondialdehyde, and glutaraldehyde.

[0800] Preferred, non-limiting examples of homobifunctional acylatingreagents include nitrophenyl esters of dicarboxylic acids.

[0801] Preferred, non-limiting examples of homobifunctional aromaticsulfonyl chlorides include phenol-2,4-disulfonyl chloride, andα-naphthol-2,4-disulfonyl chloride.

[0802] Preferred, non-limiting examples of additional amino-reactivehomobifunctional reagents include erythritolbiscarbonate which reactswith amines to give biscarbamates.

[0803] b. Homobifunctional Crosslinkers Reactive With Free SulfhydrylGroups

[0804] Synthesis, properties, and applications of such reagents aredescribed in the literature (for reviews of crosslinking procedures andreagents, see above). Many of the reagents are commercially available(e.g., Pierce Chemical Company, Rockford, Ill.; Sigma Chemical Company,St. Louis, Mo.; Molecular Probes, Inc., Eugene, Oreg.).

[0805] Preferred, non-limiting examples of homobifunctional maleimidesinclude bismaleimidohexane (BMH), N,N′-(1,3-phenylene) bismaleimide,N,N′-(1,2-phenylene)bismaleimide, azophenyldimaleimide, andbis(N-maleimidomethyl)ether.

[0806] Preferred, non-limiting examples of homobifunctional pyridyldisulfides include 1,4-di-3′-(2′-pyridyldithio)propionamidobutane(DPDPB).

[0807] Preferred, non-limiting examples of homobifunctional alkylhalides include 2,2′-dicarboxy-4,4′-diiodoacetamidoazobenzene,α,α′-diiodo-p-xylenesulfonic acid, α,α′-dibromo-p-xylenesulfonic acid,N,N′-bis(b-bromoethyl)benzylamine, N,N′-di(bromoacetyl)phenylthydrazine,and 1,2-di(bromoacetyl)amino-3-phenylpropane.

[0808] c. Homobifunctional Photoactivatable Crosslinkers

[0809] Synthesis, properties, and applications of such reagents aredescribed in the literature (for reviews of crosslinking procedures andreagents, see above). Some of the reagents are commercially available(e.g., Pierce Chemical Company, Rockford, Ill.; Sigma Chemical Company,St. Louis, Mo.; Molecular Probes, Inc., Eugene, Oreg.).

[0810] Preferred, non-limiting examples of homobifunctionalphotoactivatable crosslinker includebis-β-(4-azidosalicylamido)ethyldisulfide (BASED),di-N-(2-nitro-4-azidophenyl)-cystamine-S,S-dioxide (DNCO), and4,4′-dithiobisphenylazide.

[0811] 5. HeteroBifunctional Reagents

[0812] a. Amino-Reactive HeteroBifunctional Reagents With a PyridylDisulfide Moiety

[0813] Synthesis, properties, and applications of such reagents aredescribed in the literature (for reviews of crosslinking procedures andreagents, see above). Many of the reagents are commercially available(e.g., Pierce Chemical Company, Rockford, Ill.; Sigma Chemical Company,St. Louis, Mo.; Molecular Probes, Inc., Eugene, Oreg.).

[0814] Preferred, non-limiting examples of hetero-bifunctional reagentswith a pyridyl disulfide moiety and an amino-reactive NHS ester includeN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl6-3-(2-pyridyldithio)propionamidohexanoate (LC-SPDP), sulfosuccinimidyl6-3-(2-pyridyldithio)propionamidohexanoate (sulfo-LCSPDP),4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT),and sulfosuccinimidyl 6-α-methyl-α-(2-pyridyldithio)toluamidohexanoate(sulfo-LC-SMPT).

[0815] b. Amino-Reactive HeteroBifunctional Reagents With a MaleimideMoiety

[0816] Synthesis, properties, and applications of such reagents aredescribed in the literature. Preferred, non-limiting examples ofhetero-bifunctional reagents with a maleimide moiety and anamino-reactive NHS ester include succinimidyl maleimidylacetate (AMAS),succinimidyl 3-maleimidylpropionate (BMPS),N-γ-maleimidobutyryloxysuccinimide ester(GMBS)N-γ-maleimidobutyryloxysulfo succinimide ester (sulfo-GMBS)succinimidyl 6-maleimidylhexanoate (EMCS), succinimidyl3-maleimidylbenzoate (SMB), m-maleimidobenzoyl-N-hydroxysuccinimideester (MBS), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester(sulfo-MBS), succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC),sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), andsulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB).

[0817] c. Amino-Reactive HeteroBifunctional Reagents With an AlkylHalide Moiety

[0818] Synthesis, properties, and applications of such reagents aredescribed in the literature. Preferred, non-limiting examples ofhetero-bifunctional reagents with an alkyl halide moiety and anamino-reactive NHS ester includeN-succinimidyl-(4-iodoacetyl)aminobenzoate (SIAB),sulfosuccinimidyl-(4-iodoacetyl)aminobenzoate (sulfo-SIAB),succinimidyl-6-(iodoacetyl)aminohexanoate (SIAX),succinimidyl-6-(6-((iodoacetyl)-amino)hexanoylamino)hexanoate (SIAXX),succinimidyl-6-(((4-(iodoacetyl)-amino)-methyl)-cyclohexane-1-carbonyl)aminohexanoate(SIACX), andsuccinimidyl-4((iodoacetyl)-amino)methylcyclohexane-1-carboxylate(SIAC).

[0819] A preferred example of a hetero-bifunctional reagent with anamino-reactive NHS ester and an alkyl dihalide moiety isN-hydroxysuccinimidyl 2,3-dibromopropionate (SDBP). SDBP introducesintramolecular crosslinks to the affinity component by conjugating itsamino groups. The reactivity of the dibromopropionyl moiety towardsprimary amine groups is controlled by the reaction temperature (McKenzieet al., Protein Chem. 7: 581-592 (1988)).

[0820] Preferred, non-limiting examples of hetero-bifunctional reagentswith an alkyl halide moiety and an amino-reactive p-nitrophenyl estermoiety include p-nitrophenyl iodoacetate (NPIA).

[0821] Other cross-linking agents are known to those of skill in theart. See, for example, Pomato et al., U.S. Pat. No. 5,965,106. It iswithin the abilities of one of skill in the art to choose an appropriatecross-linking agent for a particular application.

[0822] d. Cleavable Linker Groups

[0823] In yet a further embodiment, the linker group is provided with agroup that can be cleaved to release the modifying group from the sugarresidue. Many cleavable groups are known in the art. See, for example,Jung et al., Biochem. Biophys. Acta 761: 152-162 (1983); Joshi et al.,J. Biol. Chem. 265: 14518-14525 (1990); Zarling et al., J. Immunol. 124:913-920 (1980); Bouizar et al., Eur. J. Biochem. 155: 141-147 (1986);Park et al., J. Biol. Chem. 261: 205-210 (1986); Browning et al., J.Immunol. 143: 1859-1867 (1989). Moreover a broad range of cleavable,bifunctional (both homo- and hetero-bifunctional) linker groups iscommercially available from suppliers such as Pierce.

[0824] Exemplary cleavable moieties can be cleaved using light, heat orreagents such as thiols, hydroxylamine, bases, periodate and the like.Moreover, certain preferred groups are cleaved in vivo in response tobeing endocytosed (e.g., cis-aconityl; see, Shen et al., Biochem.Biophys. Res. Commun. 102: 1048 (1991)). Preferred cleavable groupscomprise a cleavable moiety which is a member selected from the groupconsisting of disulfide, ester, imide, carbonate, nitrobenzyl, phenacyland benzoin groups.

[0825] e. Conjugation of Modified Sugars to Peptides

[0826] The modified sugars are conjugated to a glycosylated ornon-glycosylated peptide using an appropriate enzyme to mediate theconjugation. Preferably, the concentrations of the modified donorsugar(s), enzyme(s) and acceptor peptide(s) are selected such thatglycosylation proceeds until the acceptor is consumed. Theconsiderations discussed below, while set forth in the context of asialyltransferase, are generally applicable to other glycosyltransferasereactions.

[0827] A number of methods of using glycosyltransferases to synthesizedesired oligosaccharide structures are known and are generallyapplicable to the instant invention. Exemplary methods are described,for instance, WO 96/32491, Ito et al., Pure Appl. Chem. 65: 753 (1993),and U.S. Pat. Nos. 5,352,670, 5,374,541, and 5,545,553.

[0828] The present invention is practiced using a singleglycosyltransferase or a combination of glycosyltransferases. Forexample, one can use a combination of a sialyltransferase and agalactosyltransferase. In those embodiments using more than one enzyme,the enzymes and substrates are preferably combined in an initialreaction mixture, or the enzymes and reagents for a second enzymaticreaction are added to the reaction medium once the first enzymaticreaction is complete or nearly complete. By conducting two enzymaticreactions in sequence in a single vessel, overall yields are improvedover procedures in which an intermediate species is isolated. Moreover,cleanup and disposal of extra solvents and by-products is reduced.

[0829] In a preferred embodiment, each of the first and second enzyme isa glycosyltransferase. In another preferred embodiment, one enzyme is anendoglycosidase. In another preferred embodiment, one enzyme is anexoglycosidase. In an additional preferred embodiment, more than twoenzymes are used to assemble the modified glycoprotein of the invention.The enzymes are used to alter a saccharide structure on the peptide atany point either before or after the addition of the modified sugar tothe peptide.

[0830] In another embodiment, at least two of the enzymes areglycosyltransferases and the last sugar added to the saccharidestructure of the peptide is a non-modified sugar. Instead, the modifiedsugar is internal to the glycan structure and therefore need not be theultimate sugar on the glycan. In an exemplary embodiment,galactosyltransferase may catalyze the transfer of Gal-PEG fromUDP-Gal-PEG onto the glycan, followed by incubation in the presence ofST3Gal3 and CMP-SA, which serves to add a “capping” unmodified sialicacid onto the glycan (FIG. 23A).

[0831] In another embodiment, at least two of the enzymes used areglycosyltransferases, and at least two modified sugars are added to theglycan structures on the peptide. In this manner, two or more differentglycoconjugates may be added to one or more glycans on a peptide. Thisprocess generates glycan structures having two or more functionallydifferent modified sugars. In an exemplary embodiment, incubation of thepeptide with GnT-I, II and UDP-GlcNAc-PEG serves to add a GlcNAc-PEGmolecule to the glycan; incubation with galactosyltransferase andUDP-Gal then serves to add a Gal residue thereto; and, incubation withST3Gal3 and CMP-SA-Man-6-Phosphate serves to add aSA-mannose-6-phosphate molecule to the glycan. This series of reactionsresults in a glycan chain having the functional characteristics of aPEGylated glycan as well as mannose-6-phosphate targeting activity (FIG.23B).

[0832] In another embodiment, at least two of the enzymes used in thereaction are glycosyltransferases, and again, different modified sugarsare added to N-linked and O-linked glycans on the peptide. Thisembodiment is useful when two different modified sugars are to be addedto the glycans of a peptide, but when it is important to spatiallyseparate the modified sugars on the peptide from each other. Forexample, if the modified sugars comprise bulky molecules, including butnot limited to, PEG and other molecules such as a linker molecule, thismethod may be preferable. The modified sugars may be addedsimultaneously to the glycan structures on a peptide, or they may beadded sequentially. In an exemplary embodiment, incubation with ST3Gal3and CMP-SA-PEG serves to add sialic acid-PEG to the N-linked glycans,while incubation with ST3Gal1 and CMP-SA-bis Phosphonate serves to addsialic acid-Bis Phosphonate to the O-linked glycans (FIG. 23C).

[0833] In another embodiment, the method makes use of one or more exo-or endoglycosidase. The glycosidase is typically a mutant, which isengineered to form glycosyl bonds rather than rupture them. The mutantglycanase, sometimes called a glycosynthase, typically includes asubstitution of an amino acid residue for an active site acidic aminoacid residue. For example, when the endoglycanase is endo-H, thesubstituted active site residues will typically be Asp at position 130,Glu at position 132 or a combination thereof. The amino acids aregenerally replaced with serine, alanine, asparagine, or glutamine.Exoglycosidases such as transialylidase are also useful.

[0834] The mutant enzyme catalyzes the reaction, usually by a synthesisstep that is analogous to the reverse reaction of the endoglycanasehydrolysis step. In these embodiments, the glycosyl donor molecule(e.g., a desired oligo- or mono-saccharide structure) contains a leavinggroup and the reaction proceeds with the addition of the donor moleculeto a GlcNAc residue on the protein. For example, the leaving group canbe a halogen, such as fluoride. In other embodiments, the leaving groupis a Asn, or a Asn-peptide moiety. In yet further embodiments, theGlcNAc residue on the glycosyl donor molecule is modified. For example,the GlcNAc residue may comprise a 1,2 oxazoline moiety.

[0835] In a preferred embodiment, each of the enzymes utilized toproduce a conjugate of the invention are present in a catalytic amount.The catalytic amount of a particular enzyme varies according to theconcentration of that enzyme's substrate as well as to reactionconditions such as temperature, time and pH value. Means for determiningthe catalytic amount for a given enzyme under preselected substrateconcentrations and reaction conditions are well known to those of skillin the art.

[0836] The temperature at which an above-described process is carriedout can range from just above freezing to the temperature at which themost sensitive enzyme denatures. Preferred temperature ranges are about0° C. to about 55° C., and more preferably about 20° C. to about 37° C.In another exemplary embodiment, one or more components of the presentmethod are conducted at an elevated temperature using a thermophilicenzyme.

[0837] The reaction mixture is maintained for a period of timesufficient for the acceptor to be glycosylated, thereby forming thedesired conjugate. Some of the conjugate can often be detected after afew hours, with recoverable amounts usually being obtained within 24hours or less. Those of skill in the art understand that the rate ofreaction is dependent on a number of variable factors (e.g, enzymeconcentration, donor concentration, acceptor concentration, temperature,solvent volume), which are optimized for a selected system.

[0838] The present invention also provides for the industrial-scaleproduction of modified peptides. As used herein, an industrial scalegenerally produces at least one gram of finished, purified conjugate.

[0839] In the discussion that follows, the invention is exemplified bythe conjugation of modified sialic acid moieties to a glycosylatedpeptide. The exemplary modified sialic acid is labeled with PEG. Thefocus of the following discussion on the use of PEG-modified sialic acidand glycosylated peptides is for clarity of illustration and is notintended to imply that the invention is limited to the conjugation ofthese two partners. One of skill understands that the discussion isgenerally applicable to the additions of modified glycosyl moietiesother than sialic acid. Moreover, the discussion is equally applicableto the modification of a glycosyl unit with agents other than PEGincluding other water-soluble polymers, therapeutic moieties, andbiomolecules.

[0840] An enzymatic approach can be used for the selective introductionof PEGylated or PPGylated carbohydrates onto a peptide or glycopeptide.The method utilizes modified sugars containing PEG, PPG, or a maskedreactive functional group, and is combined with the appropriateglycosyltransferase or glycosynthase. By selecting theglycosyltransferase that will make the desired carbohydrate linkage andutilizing the modified sugar as the donor substrate, the PEG or PPG canbe introduced directly onto the peptide backbone, onto existing sugarresidues of a glycopeptide or onto sugar residues that have been addedto a peptide.

[0841] An acceptor for the sialyltransferase is present on the peptideto be modified by the methods of the present invention either as anaturally occurring structure or one placed there recombinantly,enzymatically or chemically. Suitable acceptors, include, for example,galactosyl acceptors such as Galβ1,4GlcNAc, Galβ1,4GalNAc,Galβ1,3GalNAc, lacto-N-tetraose, Galβ1,3GlcNAc, Galβ1,3Ara, Gal1,6GlcNAc, Galβ1,4Glc (lactose), and other acceptors known to those ofskill in the art (see, e.g., Paulson et al., J. Biol. Chem. 253:5617-5624 (1978)).

[0842] In one embodiment, an acceptor for the sialyltransferase ispresent on the peptide to be modified upon in vivo synthesis of thepeptide. Such peptides can be sialylated using the claimed methodswithout prior modification of the glycosylation pattern of the peptide.Alternatively, the methods of the invention can be used to sialylate apeptide that does not include a suitable acceptor; one first modifiesthe peptide to include an acceptor by methods known to those of skill inthe art. In an exemplary embodiment, a GalNAc residue is added by theaction of a GalNAc transferase.

[0843] In an exemplary embodiment, the galactosyl acceptor is assembledby attaching a galactose residue to an appropriate acceptor linked tothe peptide, e.g., a GlcNAc. The method includes incubating the peptideto be modified with a reaction mixture that contains a suitable amountof a galactosyltransferase (e.g., galβ1,3 or galβ1,4), and a suitablegalactosyl donor (e.g., UDP-galactose). The reaction is allowed toproceed substantially to completion or, alternatively, the reaction isterminated when a preselected amount of the galactose residue is added.Other methods of assembling a selected saccharide acceptor will beapparent to those of skill in the art.

[0844] In yet another embodiment, peptide-linked oligosaccharides arefirst “trimmed,” either in whole or in part, to expose either anacceptor for the sialyltransferase or a moiety to which one or moreappropriate residues can be added to obtain a suitable acceptor. Enzymessuch as glycosyltransferases and endoglycosidases (see, for example U.S.Pat. No. 5,716,812) are useful for the attaching and trimming reactions.A detailed discussion of “trimming” and remodeling N-linked and O-linkedglycans is provided elsewhere herein.

[0845] In the discussion that follows, the method of the invention isexemplified by the use of modified sugars having a water-soluble polymerattached thereto. The focus of the discussion is for clarity ofillustration. Those of skill will appreciate that the discussion isequally relevant to those embodiments in which the modified sugar bearsa therapeutic moiety, biomolecule or the like.

[0846] An exemplary embodiment of the invention in which a carbohydrateresidue is “trimmed” prior to the addition of the modified sugar is setforth in FIG. 14, which sets forth a scheme in which high mannose istrimmed back to the first generation biantennary structure. A modifiedsugar bearing a water-soluble polymer is conjugated to one or more ofthe sugar residues exposed by the “trimming back.” In one example, awater-soluble polymer is added via a GlcNAc moiety conjugated to thewater-soluble polymer. The modified GlcNAc is attached to one or both ofthe terminal mannose residues of the biantennary structure.Alternatively, an unmodified GlcNAc can be added to one or both of thetermini of the branched species.

[0847] In another exemplary embodiment, a water-soluble polymer is addedto one or both of the terminal mannose residues of the biantennarystructure via a modified sugar having a galactose residue, which isconjugated to a GlcNAc residue added onto the terminal mannose residues.Alternatively, an unmodified Gal can be added to one or both terminalGlcNAc residues.

[0848] In yet a further example, a water-soluble polymer is added onto aGal residue using a modified sialic acid.

[0849] Another exemplary embodiment is set forth in FIG. 15, whichdisplays a scheme similar to that shown in FIG. 14, in which the highmannose structure is “trimmed back” to the mannose from which thebiantennary structure branches. In one example, a water-soluble polymeris added via a GlcNAc modified with the polymer. Alternatively, anunmodified GlcNAc is added to the mannose, followed by a Gal with anattached water-soluble polymer. In yet another embodiment, unmodifiedGlcNAc and Gal residues are sequentially added to the mannose, followedby a sialic acid moiety modified with a water-soluble polymer.

[0850]FIG. 16 sets forth a further exemplary embodiment using a schemesimilar to that shown in FIG. 14, in which high mannose is “trimmedback” to the GlcNAc to which the first mannose is attached. The GlcNAcis conjugated to a Gal residue bearing a water-soluble polymer.Alternatively, an unmodified Gal is added to the GlcNAc, followed by theaddition of a sialic acid modified with a water-soluble sugar. In yet afurther example, the terminal GlcNAc is conjugated with Gal and theGlcNAc is subsequently fucosylated with a modified fucose bearing awater-soluble polymer.

[0851]FIG. 17 is a scheme similar to that shown in FIG. 14, in whichhigh mannose is trimmed back to the first GlcNAc attached to the Asn ofthe peptide. In one example, the GlcNAc of the GlcNAc-(Fuc)_(a) residueis conjugated with a GlcNAc bearing a water soluble polymer. In anotherexample, the GlcNAc of the GlcNAc-(Fuc)_(a) residue is modified withGal, which bears a water soluble polymer. In a still further embodiment,the GlcNAc is modified with Gal, followed by conjugation to the Gal of asialic acid modified with a water-soluble polymer.

[0852] Other exemplary embodiments are set forth in FIGS. 18-22. Anillustration of the array of reaction types with which the presentinvention may be practiced is provided in each of the aforementionedfigures.

[0853] The Examples set forth above provide an illustration of the powerof the methods set forth herein. Using the methods of the invention, itis possible to “trim back” and build up a carbohydrate residue ofsubstantially any desired structure. The modified sugar can be added tothe termini of the carbohydrate moiety as set forth above, or it can beintermediate between the peptide core and the terminus of thecarbohydrate.

[0854] In an exemplary embodiment, an existing sialic acid is removedfrom a glycopeptide using a sialidase, thereby unmasking all or most ofthe underlying galactosyl residues. Alternatively, a peptide orglycopeptide is labeled with galactose residues, or an oligosaccharideresidue that terminates in a galactose unit. Following the exposure ofor addition of the galactose residues, an appropriate sialyltransferaseis used to add a modified sialic acid. The approach is summarized inScheme 12.

[0855] In yet a further approach, summarized in Scheme 13, a maskedreactive functionality is present on the sialic acid. The maskedreactive group is preferably unaffected by the conditions used to attachthe modified sialic acid to the peptide. After the covalent attachmentof the modified sialic acid to the peptide, the mask is removed and thepeptide is conjugated with an agent such as PEG, PPG, a therapeuticmoiety, biomolecule or other agent. The agent is conjugated to thepeptide in a specific manner by its reaction with the unmasked reactivegroup on the modified sugar residue.

[0856] Any modified sugar can be used with its appropriateglycosyltransferase, depending on the terminal sugars of theoligosaccharide side chains of the glycopeptide (Table 4). As discussedabove, the terminal sugar of the glycopeptide required for introductionof the PEGylated or PPGylated structure can be introduced naturallyduring expression or it can be produced post expression using theappropriate glycosidase(s), glycosyltransferase(s) or mix ofglycosidase(s) and glycosyltransferase(s). TABLE 4 Modified sugars.

UPD-galactose-derivatives

UDP-galactosamine-derivatives (when A = NH, R₄ may be acetyl)

UDP-Glucose-derivatives

UDP-Glucosamine-derivatives (when A = NH, R₄ may be acetyl)

GDP-Mannose-derivatives

GDP-fucose-derivatives

[0857] In a further exemplary embodiment, UDP-galactose-PEG is reactedwith bovine milk β1,4-galactosyltransferase, thereby transferring themodified galactose to the appropriate terminal N-acetylglucosaminestructure. The terminal, GlcNAc residues on the glycopeptide may beproduced during expression, as may occur in such expression systems asmammalian, insect, plant or fungus, but also can be produced by treatingthe glycopeptide with a sialidase and/or glycosidase and/orglycosyltransferase, as required.

[0858] In another exemplary embodiment, a GlcNAc transferase, such asGnT-I-IV, is utilized to transfer PEGylated-GlcNc to a mannose residueon a glycopeptide. In a still further exemplary embodiment, the N-and/or O-linked glycan structures are enzymatically removed from aglycopeptide to expose an amino acid or a terminal glycosyl residue thatis subsequently conjugated with the modified sugar. For example, anendoglycanase is used to remove the N-linked structures of aglycopeptide to expose a terminal GlcNAc as a GlcNAc-linked-Asn on theglycopeptide. UDP-Gal-PEG and the appropriate galactosyltransferase isused to introduce the PEG- or PPG-galactose functionality onto theexposed GlcNAc.

[0859] In an alternative embodiment, the modified sugar is addeddirectly to the peptide backbone using a glycosyltransferase known totransfer sugar residues to the peptide backbone. This exemplaryembodiment is set forth in Scheme 14. Exemplary glycosyltransferasesuseful in practicing the present invention include, but are not limitedto, GalNAc transferases (GalNAc T1-14), GlcNAc transferases,fucosyltransferases, glucosyltransferases, xylosyltransferases,mannosyltransferases and the like. Use of this approach allows thedirect addition of modified sugars onto peptides that lack anycarbohydrates or, alternatively, onto existing glycopeptides. In bothcases, the addition of the modified sugar occurs at specific positionson the peptide backbone as defined by the substrate specificity of theglycosyltransferase and not in a random manner as occurs duringmodification of a protein's peptide backbone using chemical methods. Anarray of agents can be introduced into proteins or glycopeptides thatlack the glycosyltransferase substrate peptide sequence by engineeringthe appropriate amino acid sequence into the peptide chain.

[0860] In each of the exemplary embodiments set forth above, one or moreadditional chemical or enzymatic modification steps can be utilizedfollowing the conjugation of the modified sugar to the peptide. In anexemplary embodiment, an enzyme (e.g., fucosyltransferase) is used toappend a glycosyl unit (e.g., fucose) onto the terminal modified sugarattached to the peptide. In another example, an enzymatic reaction isutilized to “cap” sites to which the modified sugar failed to conjugate.Alternatively, a chemical reaction is utilized to alter the structure ofthe conjugated modified sugar. For example, the conjugated modifiedsugar is reacted with agents that stabilize or destabilize its linkagewith the peptide component to which the modified sugar is attached. Inanother example, a component of the modified sugar is deprotectedfollowing its conjugation to the peptide. One of skill will appreciatethat there is an array of enzymatic and chemical procedures that areuseful in the methods of the invention at a stage after the modifiedsugar is conjugated to the peptide. Further elaboration of the modifiedsugar-peptide conjugate is within the scope of the invention.

[0861] Peptide Targeting With Mannose-6-Phosphate

[0862] In an exemplary embodiment the peptide is derivatized with atleast one mannose-6-phosphate moiety. The mannose-6-phosphate moietytargets the peptide to a lysosome of a cell, and is useful, for example,to target therapeutic proteins to lysosomes for therapy of lysosomalstorage diseases.

[0863] Lysosomal storage diseases are a group of over 40 disorders whichare the result of defects in genes encoding enzymes that break downglycolipid or polysaccharide waste products within the lysosomes ofcells. The enzymatic products, e.g., sugars and lipids, are thenrecycled into new products. Each of these disorders results from aninherited autosomal or X-linked recessive trait which affects the levelsof enzymes in the lysosome. Generally, there is no biological orfunctional activity of the affected enzymes in the cells and tissues ofaffected individuals. Table 5 provides a list of representative storagediseases and the enzymatic defect associated with the diseases. In suchdiseases the deficiency in enzyme function creates a progressivesystemic deposition of lipid or carbohydrate substrate in lysosomes incells in the body, eventually causing loss of organ function and death.The genetic etiology, clinical manifestations, molecular biology andpossibility of the lysosomal storage diseases are detailed in Scriver etal., eds., THE METABOLIC AND MOLECULAR BASIS OF INHERITED DISEASE,7.sup.th Ed., Vol. 11, McGraw Hill, (1995). TABLE 5 Lysosomal storagediseases and associated enzymatic defects Disease Enzymatic Defect Pompedisease acid α-glucosidase (acid maltase) MPSI* (Hurler disease)α-L-iduronidase MPSII (Hunter disease) iduronate sulfatase MPSIII(Sanfilippo) heparan N-sulfatase MPS IV (Morquio A)galactose-6-sulfatase MPS IV (Morquio B) acid β-galactosidase MPS VII(Sly disease) β-glucoronidase I-cell disease N-acetylglucosamine-1-phosphotransferase Schindler disease α-N-acetylgalactosaminidase(α-galactosidase B) Wolman disease acid lipase Cholesterol ester storagedisease acid lipase Farber disease lysosomal acid ceramidaseNiemann-Pick disease acid sphingomyelinase Gaucher diseaseglucocerebrosidase Krabbe disease galactosylceramidase Fabry diseaseα-galactosidase A GMl gangliosidosis acid β-galactosidaseGalactosialidosis β-galactosidase and neuraminidase Tay-Sach's diseasehexosaminidase A Magakaryotic leukodystrophy arylsulphatase a Sandhoffdisease hexosaminidase A and B

[0864] De Duve first suggested that replacement of the missing lysosomalenzyme with exogenous biologically active enzyme might be a viableapproach to treatment of lysosomal storage diseases (De Duve, Fed. Proc.23: 1045 (1964). Since that time, various studies have suggested thatenzyme replacement therapy may be beneficial for treating variouslysosomal storage diseases. The best success has been shown withindividuals with type I Gaucher disease, who have been treated withexogenous enzyme (β-glucocerebrosidase), prepared from placenta(Ceredase™) or, more recently, recombinantly (Cerezyme™). It has beensuggested that enzyme replacement may also be beneficial for treatingFabry's disease, as well as other lysosomal storage diseases. See, forexample, Dawson et al., Ped. Res. 7(8): 684-690 (1973) (in vitro) andMapes et al., Science 169: 987 (1970) (in vivo). Clinical trials ofenzyme replacement therapy have been reported for Fabry patients usinginfusions of normal plasma (Mapes et al., Science 169: 987-989 (1970)),α-galactosidase A purified from placenta (Brady et al., N. Eng. J. Med.279: 1163 (1973)); or α-galactosidase A purified from spleen or plasma(Desnick et al., Proc. Natl. Acad. Sci., USA 76: 5326-5330 (1979)) andhave demonstrated the biochemical effectiveness of direct enzymereplacement for Fabry disease. These studies indicate the potential foreliminating, or significantly reducing, the pathological glycolipidstorage by repeated enzyme replacement. For example, in one study(Desnick et al., supra), intravenous injection of purified enzymeresulted in a transient reduction in the plasma levels of the storedlipid substrate, globotriasylceramide.

[0865] Accordingly, there exists a need in the art for methods forproviding sufficient quantities of biologically active lysosomalenzymes, such as human α-galactosidase A, to deficient cells. Recently,recombinant approaches have attempted to address these needs, see, e.g.,U.S. Pat. Nos. 5,658,567; 5,580,757; Bishop et al., Proc. Natl. Acad.Sci., USA. 83: 4859-4863 (1986); Medin et al., Proc. Natl. Acad. Sci.,USA. 93: 7917-7922 (1996); Novo, F. J., Gene Therapy. 4: 488-492 (1997);Ohshima et al., Proc. Natl. Acad. Sci., USA. 94: 2540-2544 (1997); andSugimoto et al., Human Gene Therapy 6: 905-915, (1995). Through themannose-6-phosphate mediated targeting of therapeutic peptides tolysosomes, the present invention provides compositions and methods fordelivering sufficient quantities of biologically active lysosomalpeptides to deficient cells.

[0866] Thus, in an exemplary embodiment, the present invention providesa peptide according to Table 7 that is derivatized withmannose-6-phosphate (FIG. 24 and FIG. 25). The peptide may berecombinantly or chemically prepared. Moreover, the peptide can be thefull, natural sequence, or it may be modified by, for example,truncation, extension, or it may include substitutions or deletions.Exemplary proteins that are remodeled using a method of the presentinvention include glucocerebrosidase, β-glucosidase, α-galactosidase A,acid-α-glucosidase (acid maltase). Representative modified peptides thatare in clinical use include, but are not limited to, Ceredase™,Cerezyme™, and Fabryzyme™. A glycosyl group on modified and clinicallyrelevant peptides may also be altered utilizing a method of theinvention. The mannose-6-phosphate is attached to the peptide via aglycosyl linking group. In an exemplary embodiment, the glycosyl linkinggroup is derived from sialic acid. Exemplary sialic acid-derivedglycosyl linking groups are set forth in Table 3, in which one or moreof the “R” moieties is mannose-6-phosphate or a spacer group having oneor more mannose-6-phosphate moieties attached thereto. The modifiedsialic acid moiety is preferably the terminal residue of anoligosaccharide linked to the surface of the peptide (FIG. 26)

[0867] In addition to the mannose-6-phosphate, the peptides of theinvention may be further derivatized with a moiety such as awater-soluble polymer, a therapeutic moiety, or an additional targetingmoiety. Methods for attaching these and other groups are set forthherein. In an exemplary embodiment, the group other thanmannose-6-phosphate is attached to the peptide via a derivatized sialicacid derivative according to Table 3, in which one or more of the “R”moieties is a group other than mannose-6-phosphate.

[0868] In an exemplary embodiment, a sialic acid moiety modified with aCbz-protected glycine-based linker arm is prepared. The correspondingnucleotide sugar is prepared and the Cbz group is removed by catalytichydrogenation. The resulting nucleotide sugar has an available, reactiveamine that is contacted with an activated mannose-6-phosphatederivative, providing a mannose-6-phosphate derivatized nucleotide sugarthat is useful in practicing the methods of the invention.

[0869] As shown in the scheme below (scheme 15), an exemplary activatedmannose-6-phosphate derivative is formed by converting a2-bromo-benzyl-protected phosphotriester into the correspondingtriflate, in situ, and reacting the triflate with a linker having areactive oxygen-containing moiety, forming an ether linkage between thesugar and the linker. The benzyl protecting groups are removed bycatalytic hydrogenation, and the methyl ester of the linker ishydrolyzed, providing the corresponding carboxylic acid. The carboxylicacid is activated by any method known in the art. An exemplaryactivation procedure relies upon the conversion of the carboxylic acidto the N-hydroxysuccinimide ester.

[0870] In another exemplary embodiment, as shown in the scheme below(scheme 16), a N-acetylated sialic acid is converted to an amine bymanipulation of the pyruvyl moiety. Thus, the primary hydroxyl isconverted to a sulfonate ester and reacted with sodium azide. The azideis catalytically reduced to the corresponding amine. The sugar issubsequently converted to its nucleotide analogue and coupled, throughthe amine group, to the linker arm-derivatized mannose-6-phosphateprepared as discussed above.

[0871] Peptides useful to treat lysosomal storage disease can bederivatized with other targeting moieties including, but not limited to,transferrin (to deliver the peptide across the blood-brain barrier, andto endosomes), carnitine (to deliver the peptide to muscle cells), andphosphonates, e.g, bisphosphonate (to target the peptide to bone andother calciferous tissues). The targeting moiety and therapeutic peptideare conjugated by any method discussed herein or otherwise known in theart.

[0872] In an exemplary embodiment, the targeting agent and thetherapeutic peptide are coupled via a linker moiety. In this embodiment,at least one of the therapeutic peptide or the targeting agent iscoupled to the linker moiety via an intact glycosyl linking groupaccording to a method of the invention. In an exemplary embodiment, thelinker moiety includes a poly(ether) such as poly(ethylene glycol). Inanother exemplary embodiment, the linker moiety includes at least onebond that is degraded in vivo, releasing the therapeutic peptide fromthe targeting agent, following delivery of the conjugate to the targetedtissue or region of the body.

[0873] In yet another exemplary embodiment, the in vivo distribution ofthe therapeutic moiety is altered via altering a glycoform on thetherapeutic moiety without conjugating the therapeutic peptide to atargeting moiety. For example, the therapeutic peptide can be shuntedaway from uptake by the reticuloendothelial system by capping a terminalgalactose moiety of a glycosyl group with sialic acid (or a derivativethereof) (FIGS. 24 and 27). Sialylation to cover terminal Gal avoidsuptake of the peptide by hepatic asialoglycoprotein (ASGP) receptors,and may extend the half life of the peptide as compared with peptideshaving only complex glycan chains, in the absence of sialylation.

[0874] II. Peptide/Glycopeptides of the Invention

[0875] In one embodiment, the present invention provides a compositioncomprising multiple copies of a single peptide having an elementaltrimannosyl core as the primary glycan structure attached thereto. Inpreferred embodiments, the peptide may be a therapeutic molecule. Thenatural form of the peptide may comprise complex N-linked glycans or maybe a high mannose glycan. The peptide may be a mammalian peptide, and ispreferably a human peptide. In some embodiments the peptide is selectedfrom the group consisting of an immunoglobulin, erythropoietin,tissue-type activator peptide, and others (See FIG. 28).

[0876] Exemplary peptides whose glycans can be remodeled using themethods of the invention are set forth in FIG. 28. TABLE 6 Preferredpeptides for glycan remodeling Hormones and Growth Factors Receptors andChimeric Receptors G-CSF CD4 GM-CSF Tumor Necrosis Factor receptor(TNF-R) TPO TNF-R: IgG Fc fusion EPO Alpha-CD20 EPO variants PSGL-1 FSHComplement HGH GlyCAM or its chimera insulin N-CAM or its chimeraalpha-TNF Monoclonal Antibodies (Immunoglobulins) Leptin MAb-anti-RSVhuman chorionic gonadotropin MAb-anti-IL-2 receptor Enzymes andInhibitors MAb-anti-CEA TPA MAb-anti-glycoprotein IIb/IIIa TPA variantsMAb-anti-EGF Urokinase MAb-anti-Her2 Factors VII, VIII, IX, X MAb-CD20DNase MAb-alpha-CD3 Glucocerebrosidase MAb-TNFα Hirudin MAb-CD4 α1antitrypsin (α1 protease MAb-PSGL-1 inhibitor) Mab-anti F protein ofRespiratory Antithrombin III Syncytial Virus Acid α-glucosidase (acidmaltase) Anti-thrombin-III α galactosidase A Cells α-L-iduronidase Redblood cells Urokinase White blood cells (e.g., T cells, B cells,Cytokines and Chimeric Cytokines dendritic cells, macrophages, NK cells,Interleukin-1 (IL-1), 1B, 2, 3, 4 neutrophils, monocytes and the like)Interferon-alpha (IFN-alpha) Stem cells IFN-alpha-2b Others IFN-betaHepatits B surface antigen (HbsAg) IFN-gamma IFN-omega Chimericdiphtheria toxin-IL-2

[0877] TABLE 7 Most preferred peptides for glycan remodelingAlpha-galactosidase A Interleukin-2 (IL-2) Alpha-L-iduronidase FactorVIII Anti-thrombin-III hrDNase Granulocyte colony Insulin stimulatingfactor (G-CSF) Hepatitis B surface protein (HbsAg) Interferon α HumanGrowth Hormone (HGH) Interferon β Human chorionic gonadotropinInterferon omega Urokinase Factor VII clotting factor TNF receptor-IgGFc fusion (Enbrel ™) Factor IX clotting factor MAb-Her-2 (Herceptin ™)Follicle Stimulating MAb-F protein of Respiratory Hormone (FSH)Erythropoietin (EPO) Syncytial Virus (Synagis ™) Granulocyte-macrophagecolony MAb-CD20 (Rituxan ™) stimulating factor (GM-CSF) MAb-TNFα(Remicade ™) Interferon γ MAb-Glycoprotein IIb/IIIa (Reopro ™) α₁protease inhibitor (α₁ antitrypsin) Tissue-type plasminogen activator(TPA) Glucocerebrosidase (Cerezyme ™)

[0878] A more detailed list of peptides useful in the invention andtheir source is provided in FIG. 28.

[0879] Other exemplary peptides that are modified by the methods of theinvention include members of the immunoglobulin family (e.g.,antibodies, MHC molecules, T cell receptors, and the like),intercellular receptors (e.g., integrins, receptors for hormones orgrowth factors and the like) lectins, and cytokines (e.g.,interleukins). Additional examples include tissue-type plasminogenactivator (TPA), renin, clotting factors such as Factor VIII and FactorIX, bombesin, thrombin, hematopoietic growth factor, colony stimulatingfactors, viral antigens, complement peptides, α1-antitrypsin,erythropoietin, P-selectin glycopeptide ligand-1 (PSGL-1),granulocyte-macrophage colony stimulating factor, anti-thrombin III,interleukins, interferons, peptides A and C, fibrinogen, herceptin™,leptin, glycosidases, among many others. This list of peptides isexemplary and should not be considered to be exclusive. Rather, as isapparent from the disclosure provided herein, the methods of theinvention are applicable to any peptide in which a desired glycanstructure can be fashioned.

[0880] The methods of the invention are also useful for modifyingchimeric peptides, including, but not limited to, chimeric peptides thatinclude a moiety derived from an immunoglobulin, such as IgG.

[0881] Peptides modified by the methods of the invention can besynthetic or wild-type peptides or they can be mutated peptides,produced by methods known in the art, such as site-directed mutagenesis.Glycosylation of peptides is typically either N-linked or O-linked. Anexemplary N-linkage is the attachment of the modified sugar to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of a carbohydrate moiety to the asparagine side chain. Thus,the presence of either of these tripeptide sequences in a peptidecreates a potential glycosylation site. As described elsewhere herein,O-linked glycosylation refers to the attachment of one sugar (e.g.,N-acetylgalactosamine, galactose, mannose, GlcNAc, glucose, fucose orxylose) to a hydroxy side chain of a hydroxyamino acid, preferablyserine or threonine, although 5-hydroxyproline or 5-hydroxylysine mayalso be used.

[0882] Several exemplary embodiments of the invention are discussedbelow. While several of these embodiments use peptides having nameshaving trademarks, and other specific peptides as the exemplary peptide,these examples are not confined to any specific peptide. The followingexemplary embodiments are contemplated to include all peptideequivalents and variants of any peptide. Such variants include, but arenot limited to, adding and deleting N-linked and O-linked glycosylationsites, and fusion proteins with added glycosylation sites. One of skillin the art will appreciate that the following embodiments and the basicmethods disclosed therein can be applied to many peptides with equalsuccess.

[0883] In one exemplary embodiment, the present invention providesmethods for modifying Granulocyte Colony Stimulating Factor (G-CSF).FIGS. 29A to 29G set forth some examples of how this is accomplishedusing the methodology disclosed herein. In FIG. 29B, a G-CSF peptidethat is expressed in a mammalian cell system is trimmed back using asialidase. The residues thus exposed are modified by the addition of asialic acid-poly(ethylene glycol) moiety (PEG moiety), using anappropriate donor therefor and ST3Gal1. FIG. 29C sets forth an exemplaryscheme for modifying a G-CSF peptide that is expressed in an insectcell. The peptide is modified by adding a galactose moiety using anappropriate donor thereof and a galactosyltransferase. The galactoseresidues are functionalized with PEG via a sialic acid-PEG derivative,through the action of ST3Gal1. In FIG. 29D, bacterially expressed G-CSFis contacted with an N-acetylgalactosamine donor andN-acetylgalactosamine transferase. The peptide is functionalized withPEG, using a PEGylated sialic acid donor and a sialyltransferase. InFIG. 29E, mammalian cell expressed G-CSF is contacted with a sialic aciddonor that is modified with levulinic acid, adding a reactive ketone tothe sialic acid donor. After addition to a glycosyl residue on theglycan on the peptide, the ketone is derivatized with a moiety such as ahydrazine- or amine-PEG. In FIG. 29F, bacterially expressed G-CSF isremodeled by contacting the peptide with an endo-GalNAc enzyme underconditions where it functions in a synthetic, rather than a hydrolyticmanner, thereby adding a PEG-Gal-GalNAc molecule from an activatedderivative thereof. FIG. 29G provides another route for remodelingbacterially expressed G-CSF. The polypeptide is derivatized with aPEGylated N-acetylgalactosamine residue by contacting the polypeptidewith an N-acetylgalactosamine transferase and an appropriate donor ofPEGylated N-acetylgalactosamine.

[0884] In another exemplary embodiment, the invention provides methodsfor modifying Interferon α-14C (IFNα14C), as shown in FIGS. 30A to 30N.The various forms of IFNα are disclosed elsewhere herein. In FIG. 30B,IFNα14C expressed in mammalian cells is first treated with sialidase totrim back the sialic acid units thereon, and then the molecule isPEGylated using ST3Gal3 and a PEGylated sialic acid donor. In FIG. 30C,N-acetylglucosamine is first added to IFNα14C which has been expressedin insect or fungal cells, where the reaction is conducted via theaction of GnT-I and/or II using an N-acetylglucosamine donor. Thepolypeptide is then PEGylated using a galactosyltransferase and a donorof PEG-galactose. In FIG. 30D, IFNα14C expressed in yeast is firsttreated with Endo-H to trim back the glycosyl units thereon. Themolecules is galactosylated using a galactosyltransferase and agalactose donor, and it is then PEGylated using ST3Gal3 and a donor ofPEG-sialic acid. In FIG. 30F, IFNα14C produced by mammalian cells ismodified to inched a PEG moiety using ST3Gal3 and a donor of PEG-sialicacid. In FIG. 30G, IFNα14C expressed in insect of fungal cells first hasN-acetylglucosamine added using one or more of GnT-I, II, IV, and V, andan N-acetylglucosamine donor. The protein is subsequently galactosylatedusing an appropriate donor and a galactosyltransferase. Then, IFNα14C isPEGylated using ST3Gal3 and a donor of PEG-sialic acid. In FIG. 30H,yeast produced IFNα14C is first treated with mannosidases to trim backthe mannosyl groups. N-acetylglucosamine is then added using a donor ofN-acetylglucosamine and one or more of GnT-I, II, IV, and V. IFNα14C isfurther galactosylated using an appropriate donor and agalactosyltransferase. Then, the polypeptide is PEGylated using ST3Gal3and a donor of PEG-sialic acid. In FIG. 30I, NSO cell expressed IFNα14Cis modified by capping appropriate terminal residues with a sialic aciddonor that is modified with levulinic acid, thereby adding a reactiveketone to the sialic acid donor. After addition to a glycosyl residue ofthe peptide, the ketone is derivatized with a moiety such as ahydrazine- or amine-PEG. In FIG. 30J, IFNα14C expressed by mammaliancells is PEGylated using a donor of PEG-sialic acid and α2,8-sialyltransferase. In FIG. 30K, IFNα14C produced by mammalian cellsis first treated with sialidase to trim back the terminal sialic acidresidues, and then the molecule is PEGylated using trans-sialidase andPEGylated sialic acid-lactose complex. In FIG. 30L, IFNα14C expressed ina mammalian system is sialylated using a donor of sialic acid and a2,8-sialyltransferase. In FIG. 30M, IFNα14C expressed in insect orfungal cells first has N-acetylglucosamine added using an appropriatedonor and GnT-I and/or II. The molecule is then contacted with agalactosyltransferase and a galactose donor that is derivatized with areactive sialic acid via a linker, so that the polypeptide is attachedto the reactive sialic acid via the linker and the galactose residue.The polypeptide is then contacted with ST3Gal3 and transferrin, and thusbecomes connected with transferrin via the sialic acid residue. In FIG.30N, IFNα14C expressed in either insect or fungal cells is first treatedwith endoglycanase to trim back the glycosyl groups, and is thencontacted with a galactosyltransferase and a galactose donor that isderivatized with a reactive sialic acid via a linker, so that thepolypeptide is attached to the reactive sialic acid via the linker andthe galactose residue. The molecule is then contacted with ST3Gal3 andtransferrin, and thus becomes connected with transferrin via the sialicacid residue.

[0885] In another exemplary embodiment, the invention provides methodsfor modifying Interferon α-2a or 2b (IFNα), as shown in FIGS. 30O to30EE. In FIG. 30P, IFNα produced in mammalian cells is first treatedwith sialidase to trim back the glycosyl units, and is then PEGylatedusing ST3Gal3 and a PEGylated sialic acid donor. In FIG. 30Q, IFNαexpressed in insect cells is first galactosylated using an appropriatedonor and a galactosyltransferase, and is then PEGylated using ST3Gal1and a PEGylated sialic acid donor. FIG. 30R offers another method forremodeling IFNα expressed in bacteria: PEGylated N-acetylgalactosamineis added to the protein using an appropriate donor andN-acetylgalactosamine transferase. In FIG. 30S, IFNα expressed inmammalian cells is modified by capping appropriate terminal residueswith a sialic acid donor that is modified with levulinic acid, adding areactive ketone to the sialic acid donor. After addition to a glycosylresidue of the peptide, the ketone is derivatized with a moiety such asa hydrazine- or amine-PEG. In FIG. 30T, IFNα expressed in bacteria isPEGylated using a modified enzyme Endo-N-acetylgalactosamidase, whichfunctions in a synthetic instead of a hydrolytic manner, and using aN-acetylgalactosamine donor derivatized with a PEG moiety. In FIG. 30U,N-acetylgalactosamine is first added IFNα using an appropriate donor andN-acetylgalactosamine transferase, and then is PEGylated using asialyltransferase and a PEGylated sialic acid donor. In FIG. 30V, IFNαexpressed in a mammalian system is first treated with sialidase to trimback the sialic acid residues, and is then PEGylated using a suitabledonor and ST3Gal1 and/or ST3Gal3. In FIG. 30W, IFNα expressed inmammalian cells is first treated with sialidase to trim back the sialicacid residues. The polypeptide is then contacted with ST3Gal1 and tworeactive sialic acid residues that are connect via a linker, so that thepolypeptide is attached to one reactive sialic acid via the linker andthe second sialic acid residue. The polypeptide is subsequentlycontacted with ST3Gal3 and transferrin, and thus becomes connected withtransferrin via the sialic acid residue. In FIG. 30Y, IFNα expressed inmammalian cells is first treated with sialidase to trim back the sialicacid residues, and is then PEGylated using ST3Gal 1 and a donor ofPEG-sialic acid. In FIG. 30Z, IFNα produced by insect cells is PEGylatedusing a galactosyltransferase and a donor of PEGylated galactose. InFIG. 30AA, bacterially expressed IFNα first has N-acetylgalactosamineadded using a suitable donor and N-acetylgalactosamine transferase. Theprotein is then PEGylated using a sialyltransferase and a donor ofPEG-sialic acid. In FIG. 30CC, IFNα expressed in bacteria is modified inanother procedure: PEGylated N-acetylgalactosamine is added to theprotein by N-acetylgalactosamine transferase using a donor of PEGylatedN-acetylgalactosamine. In FIG. 30DD, IFNα expressed in bacteria isremodeled in yet another scheme. The polypeptide is first contacted withN-acetylgalactosamine transferase and a donor of N-acetylgalactosaminethat is derivatized with a reactive sialic acid via a linker, so thatIFNα is attached to the reactive sialic acid via the linker and theN-acetylgalactosamine. IFNα is then contacted with ST3Gal3 andasialo-transferrin so that it becomes connected with transferrin via thesialic acid residue. Then, IFNα is capped with sialic acid residuesusing ST3Gal3 and a sialic acid donor. An additional method formodifying bacterially expressed IFNα is disclosed in FIG. 30EE, whereIFNα is first exposed to NHS—CO-linker-SA-CMP and is then connected to areactive sialic acid via the linker. It is subsequently conjugated withtransferrin using ST3Gal3 and transferrin.

[0886] The methods for remodeling INN omega are essentially identical tothose presented here for IFN alpha except that the attachment of theglycan to the IFN omega peptide occurs at amino acid residue 101 in SEQID NO:75. The nucleotide and amino acid sequences for IFN omega arepresented herein as SEQ ID NOS:74 and 75. Methods of making and usingIFN omega are found in U.S. Pat. Nos. 4,917,887 and 5,317,089, and in EPPatent No. 0170204-A.

[0887] In another exemplary embodiment, the invention provides methodsfor modifying Interferon β (IFN-β), as shown in FIGS. 31A to 31S. InFIG. 31B, IFN-β expressed in a mammalian system is first treated withsialidase to trim back the terminal sialic acid residues. The protein isthen PEGylated using ST3Gal3 and a donor of PEGylated sialic acid. FIG.31C is a scheme for modifying IFN-β produced by insect cells. First,N-acetylglucosamine is added to IFN-β using an appropriate donor andGnT-I and/or -II. The protein is then galactosylated using a galactosedonor and a galactosyltransferase. Finally, IFN-β is PEGylated usingST3Gal3 and a donor of PEG-sialic acid. In FIG. 3 ID, IFN-β expressed inyeast is first treated with Endo-H to trim back its glycosyl chains, andis then galactosylated using a galactose donor and agalactosyltransferase, and is then PEGylated using ST3Gal3 and a donorof PEGylated sialic acid. In FIG. 31E, IFN-β produced by mammalian cellsis modified by PEGylation using ST3Gal3 and a donor of sialic acidalready derivatized with a PEG moiety. In FIG. 31F, IFN-β expressed ininsect cells first has N-acetylglucosamine added by one or more ofGnT-I, II, IV, and V using a N-acetylglucosamine donor, and then isgalactosylated using a galactose donor and a galactosyltransferase, andis then PEGylated using ST3Gal3 and a donor of PEG-sialic acid. In FIG.31G, IFN-β expressed in yeast is first treated with mannosidases to trimback the mannosyl units, then has N-acetylglucosamine added using aN-acetylglucosamine donor and one or more of GnT-I, II, IV, and V. Theprotein is further galactosylated using a galactose donor and agalactosyltransferase, and then PEGylated using ST3Gal3 and a PEG-sialicacid donor. In FIG. 31H, mammalian cell expressed IFN-β is modified bycapping appropriate terminal residues with a sialic acid donor that ismodified with levulinic acid, adding a reactive ketone to the sialicacid donor. After addition to a glycosyl residue of the peptide, theketone is derivatized with a moiety such as a hydrazine- or amine-PEG.In FIG. 31I, IFN-β expressed in a mammalian system is PEGylated using adonor of PEG-sialic acid and a 2,8-sialyltransferase. In FIG. 31J, IFN-βexpressed by mammalian cells is first treated with sialidase to trimback its terminal sialic acid residues, and then PEGylated usingtrans-sialidase and a donor of PEGylated sialic acid. In FIG. 31K, IFN-βexpressed in mammalian cells is first treated with sialidase to trimback terminal sialic acid residues, then PEGylated using ST3Gal3 and adonor of PEG-sialic acid, and then sialylated using ST3Gal3 and a sialicacid donor. In FIG. 31L, IFN-β expressed in mammalian cells is firsttreated with sialidase and galactosidase to trim back the glycosylchains, then galactosylated using a galactose donor and anα-galactosyltransferase, and then PEGylated using ST3Gal3 or asialyltransferase and a donor of PEG-sialic acid. In FIG. 31M, IFN-βexpressed in mammalian cells is first treated with sialidase to trimback the glycosyl units. It is then PEGylated using ST3Gal3 and a donorof PEG-sialic acid, and is then sialylated using ST3Gal3 and a sialicacid donor. In FIG. 31N, IFN-β expressed in mammalian cells is modifiedby capping appropriate terminal residues with a sialic acid donor thatis modified with levulinic acid, adding a reactive ketone to the sialicacid donor. After addition to a glycosyl residue of the peptide, theketone is derivatized with a moiety such as a hydrazine- or amine-PEG.In FIG. 31O, IFN-β expressed in mammalian cells is sialylated using asialic acid donor and a 2,8-sialyltransferase. In FIG. 31Q, IFN-βproduced by insect cells first has N-acetylglucosamine added using adonor of N-acetylglucosamine and one or more of GnT-I, II, IV, and V,and is further PEGylated using a donor of PEG-galactose and agalactosyltransferase. In FIG. 31R, IFN-β expressed in yeast is firsttreated with endoglycanase to trim back the glycosyl groups, thengalactosylated using a galactose donor and a galactosyltransferase, andthen PEGylated using ST3Gal3 and a donor of PEG-sialic acid. In FIG. 3IS, IFN-β expressed in a mammalian system is first contacted withST3Gal3 and two reactive sialic acid residues connected via a linker, sothat the polypeptide is attached to one reactive sialic acid via thelinker and the second sialic acid residue. The polypeptide is thencontacted with ST3Gal3 and desialylated transferrin, and thus becomesconnected with transferrin via the sialic acid residue. Then, IFN-β isfurther sialylated using a sialic acid donor and ST3Gal3.

[0888] In another exemplary embodiment, the invention provides methodsfor modifying Factor VII or VIa, as shown in FIGS. 32A to 32D. In FIG.32B, Factor VII or VIa produced by a mammalian system is first treatedwith sialidase to trim back the terminal sialic acid residues, and thenPEGylated using ST3Gal3 and a donor of PEGylated sialic acid. FIG. 32C,Factor VII or VIIa expressed by mammalian cells is first treated withsialidase to trim back the terminal sialic acid residues, and thenPEGylated using ST3Gal3 and a donor of PEGylated sialic acid. Further,the polypeptide is sialylated with ST3Gal3 and a sialic acid donor. FIG.32D offers another modification scheme for Factor VII or VIIa producedby mammalian cells: the polypeptide is first treated with sialidase andgalactosidase to trim back its sialic acid and galactose residues, thengalactosylated using a galactosyltransferase and a galactose donor, andthen PEGylated using ST3Gal3 and a donor of PEGylated sialic acid.

[0889] In another exemplary embodiment, the invention provides methodsfor modifying Factor IX, some examples of which are included in FIGS.33A to 33G. In FIG. 33B, Factor IX produced by mammalian cells is firsttreated with sialidase to trim back the terminal sialic acid residues,and is then PEGylated with ST3Gal3 using a PEG-sialic acid donor. InFIG. 33C, Factor IX expressed by mammalian cells is first treated withsialidase to trim back the terminal sialic acid residues, it is thenPEGylated using ST3Gal3 and a PEG-sialic acid donor, and furthersialylated using ST3Gal1 and a sialic acid donor. Another scheme forremodeling mammalian cell produced Factor IX can be found in FIG. 33D.The polypeptide is first treated with sialidase to trim back theterminal sialic acid residues, then galactosylated using a galactosedonor and a galactosyltransferase, further sialylated using a sialicacid donor and ST3Gal3, and then PEGylated using a donor of PEGylatedsialic acid and ST3Gal1. In FIG. 33E, Factor IX that is expressed in amammalian system is PEGylated through the process of sialylationcatalyzed by ST3Gal3 using a donor of PEG-sialic acid. In FIG. 33F,Factor IX expressed in mammalian cells is modified by cappingappropriate terminal residues with a sialic acid donor that is modifiedwith levulinic acid, adding a reactive ketone to the sialic acid donor.After addition to a glycosyl residue of the peptide, the ketone isderivatized with a moiety such as a hydrazine- or amine-PEG. FIG. 33Gprovides an additional method of modifying Factor IX. The polypeptide,produced by mammalian cells, is PEGylated using a donor of PEG-sialicacid and a 2,8-sialyltransferase.

[0890] In another exemplary embodiment, the invention provides methodsfor modification of Follicle Stimulating Hormone (FSH). FIGS. 34A to 34Jpresent some examples. In FIG. 34B, FSH is expressed in a mammaliansystem and modified by treatment of sialidase to trim back terminalsialic acid residues, followed by PEGylation using ST3Gal3 and a donorof PEG-sialic acid. In FIG. 34C, FSH expressed in mammalian cells isfirst treated with sialidase to trim back terminal sialic acid residues,then PEGylated using ST3Gal3 and a donor of PEG-sialic acid, and thensialylated using ST3Gal3 and a sialic acid donor. FIG. 34D provides ascheme for modifying FSH expressed in a mammalian system. Thepolypeptide is treated with sialidase and galactosidase to trim back itssialic acid and galactose residues, then galactosylated using agalactose donor and a galactosyltransferase, and then PEGylated usingST3Gal3 and a donor of PEG-sialic acid. In FIG. 34E, FSH expressed inmammalian cells is modified in the following procedure: FSH is firsttreated with sialidase to trim back the sialic acid residues, thenPEGylated using ST3Gal3 and a donor of PEG-sialic acid, and is thensialylated using ST3Gal3 and a sialic acid donor. FIG. 34F offersanother example of modifying FSH produced by mammalian cells: Thepolypeptide is modified by capping appropriate terminal residues with asialic acid donor that is modified with levulinic acid, adding areactive ketone to the sialic acid donor. After addition to a glycosylresidue of the peptide, the ketone is derivatized with a moiety such asa hydrazine- or amine-PEG. In FIG. 34G, FSH expressed in a mammaliansystem is modified in another procedure: the polypeptide is remodeledwith addition of sialic acid using a sialic acid donor and an a2,8-sialyltransferase. In FIG. 34H, FSH is expressed in insect cells andmodified in the following procedure: N-acetylglucosamine is first addedto FSH using an appropriate N-acetylglucosamine donor and one or more ofGnT-I, II, IV, and V; FSH is then PEGylated using a donor ofPEG-galactose and a galactosyltransferase. FIG. 34I depicts a scheme ofmodifying FSH produced by yeast. According to this scheme, FSH is firsttreated with endoglycanase to trim back the glycosyl groups,galactosylated using a galactose donor and a galactosyltransferase, andis then PEGylated with ST3Gal3 and a donor of PEG-sialic acid. In FIG.34J, FSH expressed by mammalian cells is first contacted with ST3Gal3and two reactive sialic acid residues via a linker, so that thepolypeptide is attached to a reactive sialic acid via the linker and asecond sialic acid residue. The polypeptide is then contacted withST3Gal1 and desialylated chorionic gonadotrophin (CG) produced in CHO,and thus becomes connected with CG via the second sialic acid residue.Then, FSH is sialylated using a sialic acid donor and ST3Gal3 and/orST3Gal1.

[0891] In another exemplary embodiment, the invention provides methodsfor modifying erythropoietin (EPO), FIGS. 35A to 35AA set forth someexamples which are relevant to the remodeling of both wild-type andmutant EPO peptides. In FIG. 35B, EPO expressed in various mammaliansystems is remodeled by contacting the expressed protein with asialidase to remove terminal sialic acid residues. The resulting peptideis contacted with a sialyltransferase and a CMP-sialic acid that isderivatized with a PEG moiety. In FIG. 35C, EPO that is expressed ininsect cells is remodeled with N-acetylglucosamine, using GnT-I and/orGnT-II. Galactose is then added to the peptide, usinggalactosyltransferase. PEG group is added to the remodeled peptide bycontacting it with a sialyltransferase and a CMP-sialic acid that isderivatized with a PEG moiety. In FIG. 35D, EPO that is expressed in amammalian cell system is remodeled by removing terminal sialic acidmoieties via the action of a sialidase. The terminal galactose residuesof the N-linked glycosyl units are “capped” with sialic acid, usingST3Gal3 and a sialic acid donor. The terminal galactose residues on theO-linked glycan are functionalized with a sialic acid bearing a PEGmoiety, using an appropriate sialic acid donor and ST3Gal1. In FIG. 35E,EPO that is expressed in a mammalian cell system is remodeled byfunctionalizing the N-linked glycosyl residues with a PEG-derivatizedsialic acid moiety. The peptide is contacted with ST3Gal3 and anappropriately modified sialic acid donor. In FIG. 35F, EPO that isexpressed in an insect cell system, yeast or fungi, is remodeled byadding at least one N-acetylglucosamine residues by contacting thepeptide with a N-acetylglucosamine donor and one or more of GnT-I,GnT-II, and GnT-V. The peptide is then PEGylated by contacting it with aPEGylated galactose donor and a galactosyltransferase. In FIG. 35G, EPOthat is expressed in an insect cell system, yeast or fungi, is remodeledby the addition of at least one N-acetylglucosamine residues, using anappropriate N-acetylglucosamine donor and one or more of GnT-I, GnT-II,and GnT-V. A galactosidase that is altered to operate in a synthetic,rather than a hydrolytic manner is used to add an activated PEGylatedgalactose donor to the N-acetylglucosamine residues. In FIG. 35H, EPOthat is expressed in an insect cell system, yeast or fungi, is remodeledby the addition of at least one terminal N-acetylglucosamine-PEGresidue. The peptide is contacted with GnT-I and an appropriateN-acetlyglucosamine donor that is derivatized with a PEG moiety. In FIG.35I, EPO that is expressed in an insect cell system, yeast or fungi, isremodeled by adding one or more terminal galactose-PEG residues. Thepeptide is contacted with GnT-I and an appropriate N-acetylglucosaminedonor that is derivatized with a PEG moiety. The peptide is thencontacted with galactosyltransferase and an appropriate galactose donorthat is modified with a PEG moiety. In FIG. 35J, EPO expressed in aninsect cell system, yeast or fungi, is remodeled by the addition of onemore terminal sialic acid-PEG residues. The peptide is contacted with anappropriate N-acetylglucosamine donor and GnT-I. The peptide is furthercontacted with galactosyltransferase and an appropriate galactose donor.The peptide is then contacted with ST3Gal3 and an appropriate sialicacid donor that is derivatized with a PEG moiety. In FIG. 35K, EPOexpressed in an insect cell system, yeast or fungi, is remodeled by theaddition of terminal sialic acid-PEG residues. The peptide is contactedwith an appropriate N-acetylglucosamine donor and one or more of GnT-I,GnT-II, and GnT-V. The peptide is then contacted withgalactosyltransferase and an appropriate galactose donor. The peptide isfurther contacted with ST3Gal3 and an appropriate sialic acid donor thatis derivatized with a PEG moiety. In FIG. 35L, EPO expressed in aninsect cell system, yeast or fungi, is remodeled by the addition of oneor more terminal α2,6-sialic acid-PEG residues. The peptide is contactedwith an appropriate N-acetylglucosamine donor and one or more of GnT-I,GnT-II, and GnT-V. The peptide is further contacted withgalactosyltransferase and an appropriate galactose donor. The peptide isthen contacted with α2,6-sialyltransferase and an appropriately modifiedsialic acid donor. In FIG. 35M, EPO expressed in a mammalian cell systemis remodeled by addition of one or more terminal sialic acid-PEGresidues. The peptide is contacted with a sialidase to remove terminalsialic acid residues. The peptide is further contacted with asialyltransferase and an appropriate sialic acid donor. The peptide isfurther contacted with a sialyltransferase and an appropriate sialicacid donor that is derivatized with a PEG moiety. In FIG. 35N, EPOexpressed in a mammalian cell system is remodeled by the addition of oneor more terminal sialic acid-PEG residues. The peptide is contacted witha sialyltransferase and an appropriate sialic acid donor that isderivatized with a PEG moiety. In FIG. 35O, EPO expressed in a mammaliancell system is remodeled by the addition of one or more terminalα2,8-sialic acid-PEG residues to primarily O-linked glycans. The peptideis contacted with α2,8-sialyltransferase and an appropriate sialic aciddonor that is derivatized with a PEG moiety. In FIG. 35P, EPO expressedin a mammalian cell is remodeled by the addition of one or more terminalα2,8-sialic acid-PEG residues to O-linked and N-linked glycans. Thepeptide is contacted with α2,8-sialyltransferase and an appropriatesialic acid donor that is derivatized with a PEG moiety. In FIG. 35Q,EPO expressed in yeast or fungi is remodeled by the addition of one ormore terminal sialic acid-PEG residues. The peptide is contacted withmannosidases to remove terminal mannose residues. Next, the peptide iscontacted with GnT-I and an appropriate N-acetylglucosamine donor. Thepeptide is further contacted with galactosyltransferase and anappropriate galactose donor. The peptide is then contacted with asialyltransferase and an appropriate sialic acid donor that isderivatized with a PEG moiety. In FIG. 35R, EPO expressed in yeast orfungi is remodeled by the addition of at least one terminalN-acetylglucosamine-PEG residues. The peptide is contacted withmannosidases to remove terminal mannose residue. The peptide is thencontacted with GnT-I and an appropriate N-acetylglucosamine donor thatis derivatized with a PEG moiety. In FIG. 35S, EPO expressed in yeast orfungi is remodeled by the additon of one mor more terminal sialicacid-PEG residues. The peptide is contacted with mannosidase-I to removeα2 mannose residues. The peptide is further contacted with GnT-I and anappropriate N-acetylglucosamine donor. The peptide is then contactedwith galactosyltransferase and an appropriate galacose donor. Thepeptide is then contacted with a sialyltransferase and an appropriatesialic acid donor that is derivatized with a PEG moiety. In FIG. 35U,EPO expressed in yeast or fungi is remodeled by addition of one or moregalactose-PEG residues. The peptide is contacted with endo-H to trimback glycosyl groups. The peptide is then contacted withgalactosyltransferase and an appropriate galactose donor that isderivatized with a PEG moiety. In FIG. 35V, EPO expressed in yeast orfungi is remodeled by the addition of one or more terminal sialicacid-PEG residues. The peptide is contacted with endo-H to trim backglycosyl groups. The peptide is further contacted withgalactosyltransferase and an appropriate galactose donor. The peptide isthen contacted with a sialyltransferase and an appropriate sialic aciddonor that is derivatized with a PEG moiety. In FIG. 35W, EPO expressedin an insect cell system is remodeled by the addition of terminalgalactose-PEG residues. The peptide is contacted with mannosidases toremove terminal mannose residues. The peptide is then contacted withgalactosyltransferase and an appropriate galactose donor that isderivatized with a PEG moeity. In FIG. 35Y, a mutant EPO called “novelerythropoiesis-stimulating protein” or NESP, expressed in NSO murinemyeloma cells is remodeled by capping appropriate terminal residues witha sialic acid donor that is modified with levulinic acid, adding areactive ketone to the sialic acid donor. After addition to a glycosylresidue of the peptide, the ketone is derivatized with a moiety such asa hydrazine- or amine-PEG. In FIG. 35Z, mutant EPO, i.e. NESP, expressedin a mammalian cell system is remodeled by addition of one or moreterminal sialic acid-PEG residues. PEG is added to the glycosyl residueon the glycan using a PEG-modified sialic acid and an α2,8-sialyltransferase. In FIG. 35AA, NESP expressed in a mammalian cellsystem is remodeled by the addition of terminal sialic acid residues.The sialic acid is added to the glycosyl residue using a sialic aciddonor and an α2,8-sialyltransferase.

[0892] In another exemplary embodiment, the invention provides methodsfor modifying granulocyte-macrophage colony-stimulating factor (GM-CSF),as shown in FIGS. 36A to 36K. In FIG. 36B, GM-CSF expressed in mammaliancells is first treated with sialidase to trim back the sialic acidresidues, and then PEGylated using ST3Gal3 and a donor of PEG-sialicacid. In FIG. 36C, GM-CSF expressed in mammalian cells is first treatedwith sialidase to trim back the sialic acid residues, then PEGylatedusing ST3Gal3 and a donor of PEG-sialic acid, and then is furthersialylated using a sialic acid donor and ST3Gal1 and/or ST3Gal3. In FIG.36D, GM-CSF expressed in NSO cells is first treated with sialidase andα-galactosidase to trim back the glycosyl groups, then sialylated usinga sialic acid donor and ST3Gal3, and is then PEGylated using ST3Gal 1and a donor of PEG-sialic acid. In FIG. 36E, GM-CSF expressed inmammalian cells is first treated with sialidase to trim back sialic acidresidues, then PEGylated using ST3Gal3 and a donor of PEG-sialic acid,and then is further sialylated using ST3Gal3 and a sialic acid donor. InFIG. 36F, GM-CSF expressed in mammalian cells is modified by cappingappropriate terminal residues with a sialic acid donor that is modifiedwith levulinic acid, adding a reactive ketone to the sialic acid donor.After addition to a glycosyl residue of the peptide, the ketone isderivatized with a moiety such as a hydrazine- or amine-PEG. In FIG.36G, GM-CSF expressed in mammalian cells is sialylated using a sialicacid donor and a 2,8-sialyltransferase. In FIG. 36I, GM-CSF expressed ininsect cells is modified by addition of N-acetylglucosamine using asuitable donor and one or more of GnT-I, II, IV, and V, followed byaddition of PEGylated galactose using a suitable donor and agalactosyltransferase. In FIG. 36J, yeast expressed GM-CSF is firsttreated with endoglycanase and/or mannosidase to trim back the glycosylunits, and subsequently PEGylated using a galactosyltransferase and adonor of PEG-galactose. In FIG. 36K, GM-CSF expressed in mammalian cellsis first treated with sialidase to trim back sialic acid residues, andis subsequently sialylated using ST3Gal3 and a sialic acid donor. Thepolypeptide is then contacted with ST3Gal1 and two reactive sialic acidresidues connected via a linker, so that the polypeptide is attached toone reactive sialic acid via the linker and second sialic acid residue.The polypeptide is further contacted with ST3Gal3 and transferrin, andthus becomes connected with transferrin.

[0893] In another exemplary embodiment, the invention provides methodsfor modification of Interferon gamma (IFNγ). FIGS. 37A to 37N containsome examples. In FIG. 37B, IFNγ expressed in a variety of mammaliancells is first treated with sialidase to trim back terminal sialic acidresidues, and is subsequently PEGylated using ST3Gal3 and a donor ofPEG-sialic acid. In FIG. 37C, IFNγ expressed in a mammalian system isfirst treated with sialidase to trim back terminal sialic acid residues.The polypeptide is then PEGylated using ST3Gal3 and a donor ofPEG-sialic acid, and is further sialylated with ST3Gal3 and a donor ofsialic acid. In FIG. 37D, mammalian cell expressed IFNγ is first treatedwith sialidase and α-galactosidase to trim back sialic acid andgalactose residues. The polypeptide is then galactosylated using agalactose donor and a galactosyltransferase. Then, IFNγ is PEGylatedusing a donor of PEG-sialic acid and ST3Gal3. In FIG. 37E, IFNγ that isexpressed in a mammalian system is first treated with sialidase to trimback terminal sialic acid residues. The polypeptide is then PEGylatedusing ST3Gal3 and a donor of PEG-sialic acid, and is further sialylatedwith ST3Gal3 and a sialic acid donor. FIG. 37F describes another methodfor modifying IFNγ expressed in a mammalian system. The protein ismodified by capping appropriate terminal residues with a sialic aciddonor that is modified with levulinic acid, adding a reactive ketone tothe sialic acid donor. After addition to a glycosyl residue of thepeptide, the ketone is derivatized with a moiety such as a hydrazine- oramine-PEG. In FIG. 37G, IFNγ expressed in mammalian cells is remodeledby addition of sialic acid using a sialic acid donor and an a2,8-sialyltransferase. In FIG. 37I, IFNγ expressed in insect or fungalcells is modified by addition of N-acetylglucosamine using anappropriate donor and one or more of GnT-I, II, IV, and V. The proteinis further modified by addition of PEG moieties using a donor ofPEGylated galactose and a galactosyltransferase. FIG. 37J offers amethod for modifying IFNγ expressed in yeast. The polypeptide is firsttreated with endoglycanase to trim back the saccharide chains, and thengalactosylated using a galactose donor and a galactosyltransferase.Then, IFNγ is PEGylated using a donor of PEGylated sialic acid andST3Gal3. In FIG. 37K, IFNγ produced by mammalian cells is modified asfollows: the polypeptide is first contacted with ST3Gal3 and a donor ofsialic acid that is derivatized with a reactive galactose via a linker,so that the polypeptide is attached to the reactive galactose via thelinker and sialic acid residue. The polypeptide is then contacted with agalactosyltransferase and transferrin pre-treated with endoglycanase,and thus becomes connected with transferrin via the galactose residue.In the scheme illustrated by FIG. 37L, IFNγ, which is expressed in amammalian system, is modified via the action of ST3Gal3: PEGylatedsialic acid is transferred from a suitable donor to IFNγ. FIG. 37M is anexample of modifying IFNγ expressed in insect or fungal cells, wherePEGylation of the polypeptide is achieved by transferring PEGylatedN-acetylglucosamine from a donor to IFNγ using GnT-I and/or II. In FIG.37N, IFNγ expressed in a mammalian system is remodeled with addition ofPEGylated sialic acid using a suitable donor and an a2,8-sialyltransferase.

[0894] In another exemplary embodiment, the invention provides methodsfor modifying α₁ anti-trypsin (α₁-protease inhibitor). Some suchexamples can be found in FIGS. 38A to 38N. In FIG. 38B, α₁ anti-trypsinexpressed in a variety of mammalian cells is first treated withsialidase to trim back sialic acid residues. PEGylated sialic acidresidues are then added using an appropriate donor, such as CMP-SA-PEG,and a sialyltransferase, such as ST3Gal3. FIG. 38C demonstrates anotherscheme of α₁ anti-trypsin modification. α₁ anti-trypsin expressed in amammalian system is first treated with sialidase to trim back sialicacid residues. Sialic acid residues derivatized with PEG are then addedusing an appropriate donor and a sialyltransferase, such as ST3Gal3.Subsequently, the molecule is further modified by the addition of sialicacid residues using a sialic acid donor and ST3Gal3. Optionally,mammalian cell expressed α₁ anti-trypsin is first treated with sialidaseand α-galactosidase to trim back terminal sialic acid and α-linkagegalactose residues. The polypeptide is then galactosylated usinggalactosyltransferase and a suitable galactose donor. Further, sialicacid derivatized with PEG is added by the action of ST3Gal3 using aPEGylated sialic acid donor. In FIG. 38D, α₁ anti-trypsin expressed in amammalian system first has the terminal sialic acid residues trimmedback using sialidase. PEG is then added to N-linked glycosyl residuesvia the action of ST3Gal3, which mediates the transfer of PEGylatedsialic acid from a donor, such as CMP-SA-PEG, to α₁ anti-trypsin. Moresialic acid residues are subsequently attached using a sialic acid donorand ST3Gal3. FIG. 38E illustrates another process through which α₁anti-trypsin is remodeled. α₁ anti-trypsin expressed in mammalian cellsis modified by capping appropriate terminal residues with a sialic aciddonor that is modified with levulinic acid, adding a reactive ketone tothe sialic acid donor. After addition to a glycosyl residue of thepeptide, the ketone is derivatized with a moiety such as a hydrazine- oramine-PEG. In FIG. 38F, yet another method of α₁ anti-trypsinmodification is disclosed. α₁ anti-trypsin obtained from a mammalianexpression system is remodeled with addition of sialic acid using asialic acid donor and an a 2,8-sialyltransferase. In FIG. 38H, α₁anti-trypsin is expressed in insect or yeast cells, and remodeled by theaddition of terminal N-acetylglucosamine residues by way of contactingthe polypeptide with UDP-N-acetylglucosamine and one or more of GnT-I,II, IV, or V. Then, the polypeptide is modified with PEG moieties usinga donor of PEGylated galactose and a galactosyltransferase. In FIG. 38I,α₁ anti-trypsin expressed in yeast cells is treated first withendoglycanase to trim back glycosyl chains. It is then galactosylatedwith a galactosyltransferase and a galactose donor. Then, thepolypeptide is PEGylated using ST3Gal3 and a donor of PEG-sialic acid.In FIG. 38J, α₁ anti-trypsin is expressed in a mammalian system. Thepolypeptide is first contacted with ST3Gal3 and a donor of sialic acidthat is derivatized with a reactive galactose via a linker, so that thepolypeptide is attached to the reactive galactose via the linker andsialic acid residue. The polypeptide is then contacted with agalactosyltransferase and transferrin pre-treated with endoglycanase,and thus becomes connected with transferrin via the galactose residue.In FIG. 38L, α₁ anti-trypsin expressed in yeast is first treated withendoglycanase to trim back its glycosyl groups. The protein is thenPEGylated using a galactosyltransferase and a donor of galactose with aPEG moiety. In FIG. 38M, α₁ anti-trypsin expressed in plant cells istreated with hexosaminidase, mannosidase, and xylosidase to trim backits glycosyl chains, and subsequently modified with N-acetylglucosaminederivatized with a PEG moiety, using N-acetylglucosamine transferase anda suitable donor. In FIG. 38N, α₁ anti-trypsin expressed in mammaliancells is modified by adding PEGylated sialic acid residues using ST3Gal3and a donor of sialic acid derivatized with PEG.

[0895] In another exemplary embodiment, the invention provides methodsfor modifying glucocerebrosidase (β-glucosidase, Cerezyme™ orCeredase™), as shown in FIGS. 39A to 39K. In FIG. 39B, Cerezyme™expressed in a mammalian system is first treated with sialidase to trimback terminal sialic acid residues, and is then PEGylated using ST3Gal3and a donor of PEG-sialic acid. In FIG. 39C, Cerezyme™ expressed inmammalian cells is first treated with sialidase to trim back the sialicacid residues, then has mannose-6-phosphate group attached using ST3Gal3and a reactive sialic acid derivatized with mannose-6-phosphate, andthen is sialylated using ST3Gal3 and a sialic acid donor. Optionally,NSO cell expressed Cerezyme™ is first treated with sialidase andgalactosidase to trim back the glycosyl groups, and is thengalactosylated using a galactose donor and an α-galactosyltransferase.Then, mannose-6-phosphate moiety is added to the molecule using ST3Gal3and a reactive sialic acid derivatized with mannose-6-phosphate. In FIG.39D, Cerezyme™ expressed in mammalian cells is first treated withsialidase to trim back the sialic acid residues, it is then PEGylatedusing ST3Gal3 and a donor of PEG-sialic acid, and is then sialylatedusing ST3Gal3 and a sialic acid donor. In FIG. 39E, Cerezyme™ expressedin mammalian cells is modified by capping appropriate terminal residueswith a sialic acid donor that is modified with levulinic acid, adding areactive ketone to the sialic acid donor. After addition to a glycosylresidue of the peptide, the ketone is derivatized with a moiety such asone or more mannose-6-phosphate groups. In FIG. 39F, Cerezyme™ expressedin mammalian cells is sialylated using a sialic acid donor and a2,8-sialyltransferase. In FIG. 39H, Cerezyme™ expressed in insect cellsfirst has N-acetylglucosamine added using a suitable donor and one ormore of GnT-I, II, IV, and V, and then is PEGylated using agalactosyltransferase and a donor of PEG-galactose. In FIG. 39I,Cerezyme™ expressed in yeast is first treated with endoglycanase to trimback the glycosyl groups, then galactosylated using a galactose donorand a galactosyltransferase, and then PEGylated using ST3Gal3 and adonor of PEG-sialic acid. In FIG. 39JK, Cerezyme™ expressed in mammaliancells is first contacted with ST3Gal3 and two reactive sialic acidresidues connected via a linker, so that the polypeptide is attached toone reactive sialic acid via the linker and the second sialic acidresidue. The polypeptide is then contacted with ST3Gal3 and desialylatedtransferrin, and thus becomes connected with transferrin. Then, thepolypeptide is sialylated using a sialic acid donor and ST3Gal3.

[0896] In another exemplary embodiment, the invention provides methodsfor modifying Tissue-Type Plasminogen Activator (TPA) and its mutant.Several specific modification schemes are presented in FIGS. 40A to 40W.FIG. 40B illustrates one modification procedure: after TPA is expressedby mammalian cells, it is treated with one or more of mannosidase(s) andsialidase to trim back mannosyl and/or sialic acid residues. TerminalN-acetylglucosamine is then added by contacting the polypeptide with asuitable donor of N-acetylglucosamine and one or more of GnT-I, II, IV,and V. TPA is further galactosylated using a galactose donor and agalactosyltransferase. Then, PEG is attached to the molecule by way ofsialylation catalyzed by ST3Gal3 and using a donor of sialic acidderivatized with a PEG moiety. In FIG. 40C, TPA is expressed in insector fungal cells. The modification includes the steps of addition ofN-acetylglucosamine using an appropriate donor of N-acetylglucosamineand GnT-I and/or II; galactosylation using a galactose donor and agalactosyltransferase; and attachment of PEG by way of sialylation usingST3Gal3 and a donor of sialic acid derivatized with PEG. In FIG. 40D,TPA is expressed in yeast and subsequently treated with endoglycanase totrim back the saccharide chains. The polypeptide is further PEGylatedvia the action of a galactosyltransferase, which catalyzes the transferof a PEG-galactose from a donor to TPA. In FIG. 40E, TPA is expressed ininsect or yeast cells. The polypeptide is then treated with α- andβ-mannosidases to trim back terminal mannosyl residues. Further, PEGmoieties are attached to the molecule via transfer of PEG-galactose froma suitable donor to TPA, which is mediated by a galactosyltransferase.FIG. 40F provides a different method for modification of TPA obtainedfrom an insect or yeast system: the polypeptide is remodeled by additionof N-acetylglucosamine using a donor of N-acetylglucosamine and GnT-Iand/or II, followed by PEGylation using a galactosyltransferase and adonor of PEGylated galactose. FIG. 40G offers another scheme forremodeling TPA expressed in insect or yeast cells. TerminalN-acetylglucosamine is added using a donor of N-acetylglucosamine andGnT-I and/or II. A galactosidase that is modified to operate in asynthetic, rather than a hydrolytic manner, is utilized to add PEGylatedgalactose from a proper donor to the N-acetylglucosamine residues. InFIG. 40I, TPA expressed in a mammalian system is first treated withsialidase and galactosidase to trim back sialic acid and galactoseresidues. The polypeptide is further modified by capping appropriateterminal residues with a sialic acid donor that is modified withlevulinic acid, adding a reactive ketone to the sialic acid donor. Afteraddition to a glycosyl residue of the peptide, the ketone is derivatizedwith a moiety such as a hydrazine- or amine-PEG. In FIG. 40J, TPA, whichis expressed in a mammalian system, is remodeled following this scheme:first, the polypeptide is treated with α- and β-mannosidases to trimback the terminal mannosyl residues; sialic acid residues are thenattached to terminal galactosyl residues using a sialic acid donor andST3Gal3; further, TPA is PEGylated via the transfer of PEGylatedgalactose from a donor to a N-acetylglucosaminyl residue catalyzed by agalactosyltransferase. In FIG. 40K, TPA is expressed in a plant system.The modification procedure in this example is as follows: TPA is firsttreated with hexosaminidase, mannosidase, and xylosidase to trim backits glycosyl groups; PEGylated N-acetylglucosamine is then added to TPAusing a proper donor and N-acetylglucosamine transferase. In FIG. 40M, aTPA mutant (TNK TPA), expressed in mammalian cells, is remodeled.Terminal sialic acid residues are first trimmed back using sialidase;ST3Gal3 is then used to transfer PEGylated sialic acid from a donor toTNK TPA, such that the polypeptide is PEGylated. In FIG. 40N, TNK TPAexpressed in a mammalian system is first treated with sialidase to trimback terminal sialic acid residues. The protein is then PEGylated usingCMP-SA-PEG as a donor and ST3Gal3, and further sialylated using a sialicacid donor and ST3Gal3. In FIG. 40O, NSO cell expressed TNK TPA is firsttreated with sialidase and α-galactosidase to trim back terminal sialicacid and galactose residues. TNK TPA is then galactosylated using agalactose donor and a galactosyltransferase. The last step in thisremodeling scheme is transfer of sialic acid derivatized with PEG moietyfrom a donor to TNK TPA using a sialyltransferase such as ST3Gal3. InFIG. 40Q, TNK TPA is expressed in a mammalian system and is firsttreated with sialidase to trim back terminal sialic acid residues. Theprotein is then PEGylated using ST3Gal3 and a donor of PEGylated sialicacid. Then, the protein is sialylated using a sialic acid donor andST3Gal3. In FIG. 40R, TNK TPA expressed in a mammalian system ismodified by capping appropriate terminal residues with a sialic aciddonor that is modified with levulinic acid, adding a reactive ketone tothe sialic acid donor. After addition to a glycosyl residue of thepeptide, the ketone is derivatized with a moiety such as a hydrazine- oramine-PEG. In FIG. 40S, TNK TPA expressed in mammalian cells is modifiedvia a different method: the polypeptide is remodeled with addition ofsialic acid using a sialic acid donor and a 2,8-sialyltransferase. InFIG. 40U, TNK TPA expressed in insect cells is remodeled by addition ofN-acetylglucosamine using an appropriate donor and one or more of GnT-I,II, IV, and V. The protein is further modified by addition of PEGmoieties using a donor of PEGylated galactose and agalactosyltransferase. In FIG. 40V, TNK TPA is expressed in yeast. Thepolypeptide is first treated with endoglycanase to trim back itsglycosyl chains and then PEGylated using a galactose donor derivatizedwith PEG and a galactosyltransferase. In FIG. 40W, TNK TPA is producedin a mammalian system. The polypeptide is first contacted with ST3Gal3and a donor of sialic acid that is derivatized with a reactive galactosevia a linker, so that the polypeptide is attached to the reactivegalactose via the linker and sialic acid residue. The polypeptide isthen contacted with a galactosyltransferase and anti-TNF IG chimeraproduced in CHO, and thus becomes connected with the chimera via thegalactose residue.

[0897] In another exemplary embodiment, the invention provides methodsfor modifying Interleukin-2 (IL-2). FIGS. 41A to 41G provide someexamples. FIG. 41B provides a two-step modification scheme: IL-2produced by mammalian cells is first treated with sialidase to trim backits terminal sialic acid residues, and is then PEGylated using ST3Gal3and a donor of PEGylated sialic acid. In FIG. 41C, insect cell expressedIL-2 is modified first by galactosylation using a galactose donor and agalactosyltransferase. Subsequently, IL-2 is PEGylated using ST3Gal3 anda donor of PEGylated sialic acid. In FIG. 41D, IL-2 expressed inbacteria is modified with N-acetylgalactosamine using a proper donor andN-acetylgalactosamine transferase, followed by a step of PEGylation witha PEG-sialic acid donor and a sialyltransferase. FIG. 41E offers anotherscheme of modifying IL-2 produced by a mammalian system. The polypeptideis modified by capping appropriate terminal residues with a sialic aciddonor that is modified with levulinic acid, adding a reactive ketone tothe sialic acid donor. After addition to a glycosyl residue of thepeptide, the ketone is derivatized with a moiety such as a hydrazine- oramine-PEG. FIG. 41F illustrates an example of remodeling IL-2 expressedby E. coli. The polypeptide is PEGylated using a reactiveN-acetylgalactosamine complex derivatized with a PEG group and an enzymethat is modified so that it functions as a synthetic enzyme rather thana hydrolytic one. In FIG. 41G, IL-2 expressed by bacteria is modified byaddition of PEGylated N-acetylgalactosamine using a proper donor andN-acetylgalactosamine transferase.

[0898] In another exemplary embodiment, the invention provides methodsfor modifying Factor VIII, as shown in FIGS. 42A to 42N. In FIG. 42B,Factor VIII expressed in mammalian cells is first treated with sialidaseto trim back the sialic acid residues, and is then PEGylated usingST3Gal3 and a donor of PEG-sialic acid. In FIG. 42C, Factor VIIIexpressed in mammalian cells is first treated with sialidase to trimback the sialic acid residues, then PEGylated using ST3Gal3 and a properdonor, and is then further sialylated using ST3Gal1 and a sialic aciddonor.

[0899] In FIG. 42E, mammalian cell produced Factor VIII is modified bythe single step of PEGylation, using ST3Gal3 and a donor of PEGylatedsialic acid. FIG. 42F offers another example of modification of FactorVIII that is expressed by mammalian cells. The protein is PEGylatedusing ST3Gal 1 and a donor of PEGylated sialic acid. In FIG. 42G,mammalian cell expressed Factor VIII is remodeled following anotherscheme: it is PEGylated using a 2,8-sialyltransferase and a donor ofPEG-sialic acid. In FIG. 42I, Factor VIII produce by mammalian cells ismodified by capping appropriate terminal residues with a sialic aciddonor that is modified with levulinic acid, adding a reactive ketone tothe sialic acid donor. After addition to a glycosyl residue of thepeptide, the ketone is derivatized with a moiety such as a hydrazine- oramine-PEG. In FIG. 42J, Factor VIII expressed by mammalian cells isfirst treated with Endo-H to trim back glycosyl groups. It is thenPEGylated using a galactosyltransferase and a donor of PEG-galactose. InFIG. 42K, Factor VIII expressed in a mammalian system is firstsialylated using ST3Gal3 and a sialic acid donor, then treated withEndo-H to trim back the glycosyl groups, and then PEGylated with agalactosyltransferase and a donor of PEG-galactose. In FIG. 42L, FactorVIII expressed in a mammalian system is first treated with mannosidasesto trim back terminal mannosyl residues, then has an N-acetylglucosaminegroup added using a suitable donor and GnT-I and/or II, and then isPEGylated using a galactosyltransferase and a donor of PEG-galactose. InFIG. 42M, Factor VIII expressed in mammalian cells is first treated withmannosidases to trim back mannosyl units, then has N-acetylglucosaminegroup added using N-acetylglucosamine transferase and a suitable donor.It is further galactosylated using a galactosyltransferase and agalactose donor, and then sialylated using ST3Gal3 and a sialic aciddonor. In FIG. 42N, Factor VIII is produced by mammalian cells andmodified as follows: it is first treated with mannosidases to trim backthe terminal mannosyl groups. A PEGylated N-acetylglucosamine group isthen added using GnT-I and a suitable donor of PEGylatedN-acetylglucosamine.

[0900] In another exemplary embodiment, the invention provides methodsfor modifying urokinase, as shown in FIGS. 43A to 43M. In FIG. 43B,urokinase expressed in mammalian cells is first treated with sialidaseto trim back sialic acid residues, and is then PEGylated using ST3Gal3and a donor of PEGylated sialic acid. In FIG. 43C, urokinase expressedin mammalian cells is first treated with sialidase to trim back sialicacid residues, then PEGylated using ST3Gal3 and a donor of PEGylatedsialic acid, and then sialylated using ST3Gal3 and a sialic acid donor.Optionally, urokinase expressed in a mammalian system is first treatedwith sialidase and galactosidase to trim back glycosyl chains, thengalactosylated using a galactose donor and an α-galactosyltransferase,and then PEGylated using ST3Gal3 or sialyltransferase and a donor ofPEG-sialic acid. In FIG. 43D, urokinase expressed in mammalian cells isfirst treated with sialidase to trim back sialic acid residues, thenPEGylated using ST3Gal3 and a donor of PEG-sialic acid, and then furthersialylated using ST3Gal3 and a sialic acid donor. In FIG. 43E, urokinaseexpressed in mammalian cells is modified by capping appropriate terminalresidues with a sialic acid donor that is modified with levulinic acid,adding a reactive ketone to the sialic acid donor. After addition to aglycosyl residue of the peptide, the ketone is derivatized with a moietysuch as a hydrazine- or amine-PEG. In FIG. 43F, urokinase expressed inmammalian cells is sialylated using a sialic acid donor and a2,8-sialyltransferase. In FIG. 43H, urokinase expressed in insect cellsis modified in the following steps: first, N-acetylglucosamine is addedto the polypeptide using a suitable donor of N-acetylglucosamine and oneor more of GnT-I, II, IV, and V; then PEGylated galactose is added,using a galactosyltransferase and a donor of PEG-galactose. In FIG. 43I,urokinase expressed in yeast is first treated with endoglycanase to trimback glycosyl groups, then galactosylated using a galactose donor and agalactosyltransferase, and then PEGylated using ST3Gal3 and a donor ofPEG-sialic acid. In FIG. 43J, urokinase expressed in mammalian cells isfirst contacted with ST3Gal3 and two reactive sialic acid residues thatare connected via a linker, so that the polypeptide is attached to onereactive sialic acid via the linker and second sialic acid residue. Thepolypeptide is then contacted with ST3Gal1 and desialylated urokinaseproduced in mammalian cells, and thus becomes connected with a secondmolecule of urokinase. Then, the whole molecule is further sialylatedusing a sialic donor and ST3Gal1 and/or ST3Gal3. In FIG. 43K, isolatedurokinase is first treated with sulfohydrolase to remove sulfate groups,and is then PEGylated using a sialyltransferase and a donor ofPEG-sialic acid. In FIG. 43LM, isolated urokinase is first treated withsulfohydrolase and hexosaminidase to remove sulfate groups andhexosamine groups, and then PEGylated using a galactosyltransferase anda donor of PEG-galactose.

[0901] In another exemplary embodiment, the invention provides methodsfor modifying DNase I, as shown in FIGS. 44A to 44J. In FIG. 44B, DNaseI is expressed in a mammalian system and modified in the followingsteps: first, the protein is treated with sialidase to trim back thesialic acid residues; then the protein is PEGylated with ST3Gal3 using adonor of PEG-sialic acid. In FIG. 44C, DNase I expressed in mammaliancells is first treated with sialidase to trim back the sialic acidresidues, then PEGylated with ST3Gal3 using a PEG-sialic acid donor, andis then sialylated using ST3Gal3 and a sialic acid donor. Optionally,DNase I expressed in a mammalian system is first exposed to sialidaseand galactosidase to trim back the glycosyl groups, then galactosylatedusing a galactose donor and an α-galactosyltransferase, and thenPEGylated using ST3Gal3 or sialyltransferase and a donor of PEG-sialicacid. In FIG. 44D, DNase I expressed in mammalian cells is first treatedwith sialidase to trim back the sialic acid residues, then PEGylatedusing ST3Gal3 and a PEG-sialic acid donor, and then sialylated withST3Gal3 using a sialic acid donor. In FIG. 44E, DNase I expressed inmammalian cells is modified by capping appropriate terminal residueswith a sialic acid donor that is modified with levulinic acid, adding areactive ketone to the sialic acid donor. After addition to a glycosylresidue of the peptide, the ketone is derivatized with a moiety such asa hydrazine- or amine-PEG. In FIG. 44F, DNase I expressed in mammaliancells is sialylated using a sialic acid donor and a2,8-sialyltransferase. In FIG. 44H, DNase I expressed in insect cellsfirst has N-acetylglucosamine added using a suitable donor and one ormore of GnT-I, II, IV, and V. The protein is then PEGylated using agalactosyltransferase and a donor of PEG-galactose. In FIG. 44I, DNase Iexpressed in yeast is first treated with endoglycanase to trim back theglycosyl units, then galactosylated using a galactose donor and agalactosyltransferase, and then PEGylated using ST3Gal3 and a donor ofPEG-sialic acid. In FIG. 44JK, DNase I expressed in mammalian cells isfirst contacted with ST3Gal3 and two reactive sialic acid residuesconnected via a linker, so that the polypeptide is attached to onereactive sialic acid via the linker and the second sialic acid residue.The polypeptide is then contacted with ST3Gal1 and desialylatedα-1-protease inhibitor, and thus becomes connected with the inhibitorvia the sialic acid residue. Then, the polypeptide is further sialylatedusing a suitable donor and ST3Gal 1 and/or ST3Gal3.

[0902] In another exemplary embodiment, the invention provides methodsfor modifying insulin that is mutated to contain an N-glycosylationsite, as shown in FIGS. 45A to 45L. In FIG. 45B, insulin expressed in amammalian system is first treated with sialidase to trim back the sialicacid residues, and then PEGylated using ST3Gal3 and a PEG-sialic aciddonor. In FIG. 45C, insulin expressed in insect cells is modified byaddition of PEGylated N-acetylglucosamine using an appropriate donor andGnT-I and/or II. In FIG. 45D, insulin expressed in yeast is firsttreated with Endo-H to trim back the glycosyl groups, and then PEGylatedusing a galactosyltransferase and a donor of PEG-galactose. In FIG. 45F,insulin expressed in mammalian cells is first treated with sialidase totrim back the sialic acid residues and then PEGylated using ST3Gal1 anda donor of PEG-sialic acid. In FIG. 45G, insulin expressed in insectcells is modified by means of addition of PEGylated galactose using asuitable donor and a galactosyltransferase. In FIG. 45H, insulinexpressed in bacteria first has N-acetylgalactosamine added using aproper donor and N-acetylgalactosamine transferase. The polypeptide isthen PEGylated using a sialyltransferase and a donor of PEG-sialic acid.In FIG. 45J, insulin expressed in bacteria is modified through adifferent method: PEGylated N-acetylgalactosamine is added to theprotein using a suitable donor and N-acetylgalactosamine transferase. InFIG. 45K, insulin expressed in bacteria is modified following anotherscheme: the polypeptide is first contacted with N-acetylgalactosaminetransferase and a reactive N-acetylgalactosamine that is derivatizedwith a reactive sialic acid via a linker, so that the polypeptide isattached to the reactive sialic acid via the linker andN-acetylgalactosamine. The polypeptide is then contacted with ST3Gal3and asialo-transferrin, and therefore becomes connected withtransferrin. Then, the polypeptide is sialylated using ST3Gal3 and asialic acid donor. In FIG. 45L, insulin expressed in bacteria ismodified using yet another method: the polypeptide is first exposed toNHS-CO-linker-SA-CMP and becomes connected to the reactive sialic acidresidue via the linker. The polypeptide is then conjugated totransferrin using ST3Gal3 and asialo-transferrin. Then, the polypeptideis further sialylated using ST3Gal3 and a sialic acid donor.

[0903] In another exemplary embodiment, the invention provides methodsfor modifying Hepatitis B antigen (M antigen-preS2 and S), as shown inFIGS. 46A to 46K. In FIG. 46B, M-antigen is expressed in a mammaliansystem and modified by initial treatment of sialidase to trim back thesialic acid residues and subsequent conjugation with lipid A, usingST3Gal3 and a reactive sialic acid linked to lipid A via a linker. InFIG. 46C, M-antigen expressed in mammalian cells is first treated withsialidase to trim back the terminal sialic acid residues, thenconjugated with tetanus toxin via a linker using ST3Gal1 and a reactivesialic acid residue linked to the toxin via the linker, and thensialylated using ST3Gal3 and a sialic acid donor. In FIG. 46D, M-antigenexpressed in a mammalian system is first treated with a galactosidase totrim back galactosyl residues, and then sialylated using ST3Gal3 and asialic acid donor. The polypeptide then has sialic acid derivatized withKLH added using ST3Gal1 and a suitable donor. In FIG. 46E, yeastexpressed M-antigen is first treated with a mannosidase to trim back themannosyl residues, and then conjugated to a diphtheria toxin using GnT-Iand a donor of N-acetylglucosamine linked to the diphtheria toxin. InFIG. 46F, mammalian cell expressed M-antigen is modified by cappingappropriate terminal residues with a sialic acid donor that is modifiedwith levulinic acid, adding a reactive ketone to the sialic acid donor.After addition to a glycosyl residue of the peptide, the ketone isderivatized with a moiety such as a hydrazine- or amine-PEG. In FIG.46G, M-antigen obtained from a mammalian system is remodeled bysialylation using a sialic acid donor and poly a 2,8-sialyltransferase.In FIG. 46I, M-antigen expressed in insect cells is conjugated to aNeisseria protein by using GnT-II and a suitable donor ofN-acetylglucosamine linked to the Neisseria protein. In FIG. 46J, yeastexpressed M-antigen is first treated with endoglycanase to trim back itsglycosyl chains, and then conjugated to a Neisseria protein using agalactosyltransferase and a proper donor of galactose linked to theNeisseria protein. FIG. 46K is another example of modification ofM-antigen expressed in yeast. The polypeptide is first treated withmannosidases to trim back terminal mannosyl residues, and then hasN-acetylglucosamine added using GnT-I and/or II. Subsequently, thepolypeptide is galactosylated using a galactose donor and agalactosyltransferase, and then capped with sialic acid residues using asialyltransferase and a sialic acid donor.

[0904] In another exemplary embodiment, the invention provides methodsfor modifying human growth hormone (N, V, and variants thereof), asshown in FIGS. 47A to 47K. In FIG. 47B, human growth hormone eithermutated to contain a N-linked site, or a naturally occurring isoformthat has an N-linked side (i.e., the placental enzyme) produced bymammalian cells is first treated with sialidase to trim back terminalsialic acid residues and subsequently PEGylated with ST3Gal3 and using adonor of PEGylated sialic acid. In FIG. 47C, human growth hormoneexpressed in insect cells is modified by addition of PEGylatedN-acetylglucosamine using GnT-I and/or II and a proper donor ofPEGylated N-acetylglucosamine. In FIG. 47D, human growth hormone isexpressed in yeast, treated with Endo-H to trim back glycosyl groups,and further PEGylated with a galactosyltransferase using a donor ofPEGylated galactose. In FIG. 47F, human growth hormone-mucin fusionprotein expressed in a mammalian system is modified by initial treatmentof sialidase to trim back sialic acid residues and subsequent PEGylationusing a donor of PEG-sialic acid and ST3Gal1. In FIG. 47G, human growthhormone-mucin fusion protein expressed in insect cells is remodeled byPEGylation with a galactosyltransferase and using a donor of PEGylatedgalactose. In FIG. 47H, human growth hormone-mucin fusion protein isproduced in bacteria. N-acetylgalactosamine is first added to the fusionprotein by the action of N-acetylgalactosamine transferase using a donorof N-acetylgalactosamine, followed by PEGylation of the fusion proteinusing a donor of PEG-sialic acid and a sialyltransferase. FIG. 47Idescribes another scheme of modifying bacterially expressed human growthhormone-mucin fusion protein: the fusion protein is PEGylated throughthe action of N-acetylgalactosamine transferase using a donor ofPEGylated N-acetylgalactosamine. FIG. 47J provides a further remodelingscheme for human growth hormone-mucin fusion protein. The fusion proteinis first contacted with N-acetylgalactosamine transferase and a donor ofN-acetylgalactosamine that is derivatized with a reactive sialic acidvia a linker, so that the fusion protein is attached to the reactivesialic acid via the linker and N-acetylgalactosamine. The fusion proteinis then contacted with a sialyltransferase and asialo-transferrin, andthus becomes connected with transferrin via the sialic acid residue.Then, the fusion protein is capped with sialic acid residues usingST3Gal3 and a sialic acid donor. In FIG. 47K, yet another scheme isgiven for modification of human growth hormone (N) produced in bacteria.The polypeptide is first contacted with NHS—CO-linker-SA-CMP and becomescoupled with the reactive sialic acid through the linker. Thepolypeptide is then contacted with ST3Gal3 and asialo-transferrin andbecomes linked to transferrin via the sialic acid residue. Then, thepolypeptide is sialylated using ST3Gal3 and a sialic acid donor.

[0905] In another exemplary embodiment, the invention provides methodsfor remodeling TNF receptor IgG fusion protein (TNFR-IgG, or Enbrel™),as shown in FIGS. 48A to 48G. FIG. 48B illustrates a modificationprocedure in which TNFR-IgG, expressed in a mammalian system is firstsialylated with a sialic acid donor and a sialyltransferase, ST3Gal1;the fusion protein is then galactosylated with a galactose donor and agalactosyltransferase; then, the fusion protein is PEGylated via theaction of ST3Gal3 and a donor of sialic acid derivatized with PEG. InFIG. 48C, TNFR-IgG expressed in mammalian cells is initially treatedwith sialidase to trim back sialic acid residues. PEG moieties aresubsequently attached to TNFR-IgG by way of transferring PEGylatedsialic acid from a donor to the fusion protein in a reaction catalyzedby ST3Gal1. In FIG. 48D, TNFR-IgG is expressed in a mammalian system andmodified by addition of PEG through the galactosylation process, whichis mediated by a galactosyltransferase using a PEG-galactose donor. InFIG. 48E, TNFR-IgG is expressed in a mammalian system. The first step inremodeling of the fusion protein is adding O-linked sialic acid residuesusing a sialic acid donor and a sialyltransferase, ST3Gal 1.Subsequently, PEGylated galactose is added to the fusion protein using agalactosyltransferase and a suitable donor of galactose with a PEGmoiety. In FIG. 48F, TNFR-IgG expressed in mammalian cells is modifiedfirst by capping appropriate terminal residues with a sialic acid donorthat is modified with levulinic acid, adding a reactive ketone to thesialic acid donor. After addition to a glycosyl residue of the fusionprotein, the ketone is derivatized with a moiety such as a hydrazine- oramine-PEG. In FIG. 48G, TNFR-IgG expressed in mammalian cells isremodeled by 2,8-sialyltransferase, which catalyzes the reaction inwhich PEGylated sialic acid is transferred to the fusion protein from adonor of sialic acid with a PEG moiety.

[0906] In another exemplary embodiment, the invention provides methodsfor generating Herceptin™ conjugates, as shown in FIGS. 49A to 49D. InFIG. 49B, Herceptin™ is expressed in a mammalian system and is firstgalactosylated using a galactose donor and a galactosyltransferase.Herceptin™ is then conjugated with a toxin via a sialic acid through theaction of ST3Gal3 using a reactive sialic acid-toxin complex. In FIG.49C, Herceptin™ produced in either mammalian cells or fungi isconjugated to a toxin through the process of galactosylation, using agalactosyltransferase and a reactive galactose-toxin complex. FIG. 49Dcontains another scheme of making Herceptin™ conjugates: Herceptin™produced in fungi is first treated with Endo-H to trim back glycosylgroups, then galactosylated using a galactose donor and agalactosyltransferase, and then conjugated with a radioisotope by way ofsialylation, by using ST3Gal3 and a reactive sialic acid-radioisotopecomplex. Alternatively, the reactive sialic acid moiety may haveattached only the chelating moiety can then be loaded with radioisotopeat a subsequent stage.

[0907] In another exemplary embodiment, the invention provides methodsfor making Synagis™ conjugates, as shown in FIGS. 50A to 50D. In FIG.50B, Synagis™ expressed in mammalian cells is first galactosylated usinga galactose donor and a galactosyltransferase, and then PEGylated usingST3Gal3 and a donor of PEG-sialic acid. In FIG. 50C, Synagis™ expressedin mammalian or fungal cells is PEGylated using a galactosyltransferaseand a donor of PEG-galactose. In FIG. 50D, Synagis™ expressed in firsttreated with Endo-H to trim back the glycosyl groups, thengalactosylated using a galactose donor and a galactosyltransferase, andis then PEGylated using ST3Gal3 and a donor of PEG-sialic acid.

[0908] In another exemplary embodiment, the invention provides methodsfor generating Remicade™ conjugates, as shown in FIGS. 51A to 51D. InFIG. 51B, Remicade™ expressed in a mammalian system is firstgalactosylated using a galactose donor and a galactosyltransferase, andthen PEGylated using ST3Gal3 and a donor of PEG-sialic acid. In FIG.51C, Remicade™ expressed in a mammalian system is modified by additionof PEGylated galactose using a suitable donor and agalactosyltransferase. In FIG. 51D, Remicade™ expressed in fungi isfirst treated with Endo-H to trim back the glycosyl chains, thengalactosylated using a galactose donor and a galactosyltransferase, andthen conjugated to a radioisotope using ST3Gal3 and a reactive sialicacid derivatized with the radioisotope.

[0909] In another exemplary embodiment, the invention provides methodsfor modifying Reopro, which is mutated to contain an N glycosylationsite. FIGS. 52A to 52L contain such examples. In FIG. 52B, Reoproexpressed in a mammalian system is first treated with sialidase to trimback the sialic acid residues, and then PEGylated using ST3Gal3 and adonor of PEG-sialic acid. In FIG. 52C, Reopro expressed in insect cellsis modified by addition of PEGylated N-acetylglucosamine using anappropriate donor and GnT-I and/or II. In FIG. 52D, Reopro expressed inyeast is first treated with Endo-H to trim back the glycosyl groups.Subsequently, the protein is PEGylated using a galactosyltransferase anda donor of PEG-galactose. In FIG. 52F, Reopro expressed in mammaliancells is first treated with sialidase to trim back the sialic acidresidues and then PEGylated with ST3Gal1 using a donor of PEGylatedsialic acid. In FIG. 52G, Reopro expressed in insect cells is modifiedby PEGylation using a galactosyltransferase and a donor ofPEG-galactose. In FIG. 52H, Reopro expressed in bacterial first hasN-acetylgalactosamine added using N-acetylgalactosamine transferase anda suitable donor. The protein is then PEGylated using asialyltransferase and a donor of PEG-sialic acid. In FIG. 52J, Reoproexpressed in bacteria is modified in a different scheme: it is PEGylatedvia the action of N-acetylgalactosamine transferase, using a donor ofPEGylated N-acetylgalactosamine. In FIG. 52K, bacterially expressedReopro is modified in yet another method: first, the polypeptide iscontacted with N-acetylgalactosamine transferase and a donor ofN-acetylgalactosamine that is derivatized with a reactive sialic acidvia a linker, so that the polypeptide is attached to the reactive sialicacid via the linker and N-acetylgalactosamine. The polypeptide is thencontacted with ST3Gal3 and asialo-transferrin and thus becomes connectedwith transferrin via the sialic acid residue. Then, the polypeptide iscapped with sialic acid residues using a proper donor and ST3Gal3. FIG.52L offers an additional scheme of modifying bacterially expressedReopro. The polypeptide is first exposed to NHS—CO-linker-SA-CMP andbecomes connected with the reactive sialic acid through the linker. Thepolypeptide is then contacted with ST3Gal3 and asialo-transferrin andthus becomes connected with transferrin via the sialic acid residue.Then, the polypeptide is capped with sialic acid residues using a properdonor and ST3Gal3.

[0910] In another exemplary embodiment, the invention provides methodsfor producing Rituxan™ conjugates. FIGS. 53A to 53G presents someexamples. In FIG. 53B, Rituxan™ expressed in various mammalian systemsis first galactosylated using a proper galactose donor and agalactosyltransferase. The peptide is then functionalized with a sialicacid derivatized with a toxin moiety, using a sialic acid donor andST3Gal3. In FIG. 53C, Rituxan™ expressed in mammalian cells or fungalcells is galactosylated using a galactosyltransferase and a galactosedonor, which provides the peptide galactose containing a drug moiety.FIG. 53D provides another example of remodeling Rituxan™ expressed in afungal system. The polypeptide's glycosyl groups are first trimmed backusing Endo-H. Galactose is then added using a galactosyltransferase anda galactose donor. Subsequently, a radioisotope is conjugated to themolecule through a radioisotope-complexed sialic acid donor and asialyltransferase, ST3Gal3. In FIG. 53F, Rituxan™ is expressed in amammalian system and first galactosylated using a galactosyltransferaseand a proper galactose donor; sialic acid with a PEG moiety is thenattached to the molecule using ST3Gal3 and a PEGylated sialic aciddonor. As shown in FIG. 53G, Rituxan™ expressed in fungi, yeast, ormammalian cells can also be modified in the following process: first,the polypeptide is treated with α- and β-mannosidases to remove terminalmannosyl residues; GlcNAc is then attached to the molecule using GnT-I,II and a GlcNAc donor, radioisotope is then attached by way ofgalactosylation using a galactosyltransferase and a donor of galactosethat is coupled to a chelating moiety capable of binding a radioisotope.

[0911] In another exemplary embodiment, the invention provides methodsfor modifying anti-thrombin III (AT III). FIGS. 54A to 54O present someexamples. In FIG. 54B, anti-thrombin III expressed in various mammaliansystems is remodeled by the addition of one or more terminal sialicacid-PEG moieties. The AT III molecule is first contacted with sialidaseto remove terminal sialic acid moieties. Then, the molecule is contactedwith a sialyltransferase and an appropriate sialic acid donor that hasbeen derivatized with a PEG moiety. In FIG. 54C, AT III expressed invarious mammalian systems is remodeled by the addition of sialicacid-PEG moieties. The AT III molecule is contacted with sialidase toremove terminal sialic acid moieties. The molecule is then contactedwith a ST3Gal3 and an appropriate sialic acid donor that has beenderivatized with a PEG moiety at 1.2 mol eq. The molecule is thencontacted with a ST3Gal3 and an appropriate sialic acid donor to capremaining terminal galactose moieties. In FIG. 54D, AT III is expressedin NSO murine myeloma cells is remodeled to have complex glycanmolecules with terminal sialic acid-PEG moieties. The AT III molecule iscontacted with sialidase and α-galactosidase to remove terminal sialicacid and galactose moieties. The molecule is then contacted withgalactosyltransferase and an appropriated galactose donor. The moleculeis then contacted with a ST3Gal3 and an appropriate sialic acid donorthat has been derivatized with a PEG moiety. In FIG. 54E, AT IIIexpressed in various mammalian systems is remodeled to have nearlycomplete terminal sialic acid-PEG moieties. The AT III molecule iscontacted with sialidase to remove terminal sialic acid moieties. Themolecule is then contacted with a ST3Gal3 and an appropriate sialic aciddonor that has been derivatized with a PEG moiety at 16 mol eq. Themolecule is then contacted with ST3Gal3 and an appropriate sialic aciddonor to cap remaining terminal galactose moieties. In FIG. 54F, AT IIIexpressed in various mammalian systems is remodeled by the addition ofone or more terminal sialic acid PEG moieties. The AT III molecule iscontacted with ST3Gal3 and an appropriate sialic acid donor that hasbeen derivatized with a levulinate moiety. The molecule is thencontacted with hydrazine-PEG. In FIG. 54G, AT III expressed in variousmammalian systems is remodeled by the addition of one or more terminalpoly-α2,8-linked sialic acid moieties. The AT III molecule is contactedwith poly-α2,8-sialyltransferase and an appropriate sialic acid donor.In FIG. 54I, AT III expressed in insect, yeast or fungi cells isremodeled by the addition of branching N-N-acetylglucosamine-PEGmoieties. The AT III molecule is contacted with GnT-I and an appropriateN-acetylglucosamine donor that has been derivatized with PEG. In FIG.54J, AT III expressed in yeast is remodeled by removing high mannoseglycan structures and the addition of terminal sialic acid-PEG moieties.The AT III molecule is contacted with endoglycanase to trim backglycosyl groups. The molecule is then contacted withgalactosyltransferase and an appropriate galactose donor. The moleculeis then contacted with ST3Gal3 and an appropriate sialic acid donor thathas been derivatized with a PEG moiety. In FIG. 54K, AT III expressed invarious mammalian systems is remodeled by the addition ofglycoconjugated transferrin. The AT III molecule is contacted withST3Gal3 and an appropriate sialic acid donor that has been derivatizedwith a linker-galactose donor moiety. The molecule is then contactedwith galactosyltransferase and endoglycanase-treated transferrin. InFIG. 54M, AT III expressed in yeast is remodeled by the removal ofmannose glycan structures and the addition of terminal galactose-PEGmoieties. The molecule is contacted with endoglycanase to trim backglycosyl groups. The molecule is further contacted withgalactosyltransferase and an appropriate galactose donor that has beenderivatized with a PEG moiety. In FIG. 54N, AT III expressed in plantcells is remodeled by converting the glycan structures intomammalian-type complex glycans and then adding one or more terminalgalactose-PEG moieties. The AT III molecule is contacted with xylosidaseto remove xylose residues. The molecule is then contacted withgalactosyltransferase and an appropriate galactose donor that has beenderivatized with a PEG moiety. In FIG. 54O, AT III expressed in variousmammalian systems is remodeled by the addition of one or more terminalsialic acid-PEG moieties to terminal galactose moieties. The AT IIImolecule is contacted with ST3Gal3 and an appropriate sialic acid PEGdonor that has been derivatized with PEG.

[0912] In another exemplary embodiment, the invention provides methodsfor modifying the α and β subunits of human Chorionic Gonadotropin(hCG). FIGS. 55A to 55J present some examples. In FIG. 55B, hCGexpressed in various mammalian and insect systems is remodeled by theaddition of terminal sialic acid-PEG moieties. The hCG molecule iscontacted with sialidase to remove terminal sialic acid moieties. Themolecule is then contacted with ST3Gal3 and an appropriate sialic aciddonor molecule that has been derivatized with a PEG moiety. In FIG. 55C,hCG expressed in insect cell, yeast or fungi systems is remodeled bybuilding out the N-linked glycans and the addition of terminal sialicacid-PEG moieties. The hCG molecule is contacted with GnT-I and GnT-II,and an appropriated N-acetylglucosamine donor. The molecule is thencontacted with galactosyltransferase and an appropriate galactose donor.The molecule is further contacted with ST3Gal3 and an appropriate sialicacid donor that has been derivatized with a PEG moiety. In FIG. 55D, hCGexpressed in various mammalian and insect systems is remodeled by theaddition of one or more terminal sialic acid-PEG moieties on O-linkedglycan structures. The hCG molecule is contacted with sialidase toremove terminal sialic acid moieties. The molecule is then contactedwith ST3Gal3 and an appropriate sialic acid donor to cap the glycanstructures with sialic acid moieties. The molecule is then contactedwith ST3Gal1 and an appropriate sialic acid donor that has beenderivatized with PEG. In FIG. 55E, hCG expressed in various mammalianand insect systems is remodeled by the addition of sialic acid-PEGmoieties to N-linked glycan structures. The hCG molecule is contactedwith ST3Gal3 and an appropriate sialic acid donor that has beenderivatized with PEG. In FIG. 55F, hCG expressed in insect cells, yeastor fungi, is remodeled by the addition of terminalN-acetylglucosamine-PEG molecules. The hCG molecule is contacted withGnT-I and GnT-II, and an appropriate N-acetylglucosamine donor that hasbeen derivatized with PEG. In FIG. 55G, hCG expressed in insect cells,yeast or fungi, is remodeled by the addition of not more than oneN-acetylglucosamine-PEG moiety per N-linked glycan structure. The hCGmolecule is contacted with GnT-I and an appropriate N-acetylglucosaminedonor that has been derivatized with a PEG moiety. In FIG. 55H, hCGexpressed in various mammalian systems is remodeled by the addition ofone or more terminal sialic acid-PEG moiety to O-linked glycanstructures. The hCG molecule is contacted with ST3Gal3 and anappropriate sialic acid donor that has been derivatized with PEG. InFIG. 55I, hCG expressed in various mammalian systems is remodeled by theaddition of terminal sialic acid-PEG moieties. The hCG molecule iscontacted with α2,8-SA and an appropriate sialic acid donor that hasbeen derivatized with a PEG moiety. In FIG. 55J, hCG expressed invarious mammalian systems is remodeled by the addition of terminalsialic acid moieties. The hCG molecule is contacted withpoly-alpha2,8-ST and an appropriate sialic acid donor that has beenderivatized with a PEG moiety.

[0913] In another exemplary embodiment, the invention provides methodsfor modifying alpha-galactosidase A (Fabrazyme™). FIGS. 56A to 56Jpresent some examples. In FIG. 56B, alpha-galactosidase A expressed inand secreted from various mammalian and insect systems is remodeled bythe addition of one or more terminal galactose-PEG-transferrin moieties.The alpha-galactosidase A molecule is contacted with Endo-H to trim backglycosyl groups. The molecule is then contacted withgalactosyltransferase and an appropriate galactose donor that has beenderivatized with PEG and transferrin. In FIG. 56C, alpha-galactosidase Aexpressed in and secreted from various mammal and insect cell systems isremodeled by the addition of one or more terminal sialicacid-linker-mannose-6-phosphate moieties. The alpha-galactosidase Amolecule is contacted with sialidase to remove terminal sialic acidmoieties. The molecule is further contacted with ST3Gal3 and anappropriate sialic acid donor that has been conjugated via a linker tomannose-6-phosphate. In FIG. 56D, alpha-galactosidase A expressed in NSOmurine myeloma cells is remodeled by the addition of terminal sialicacid-linker-mannose-6-phosphate moieties. The alpha-galactosidase Amolecule is contacted with sialidase and α-galactosidase to removeterminal sialic acid and galactose moieties. The molecule is thencontacted with galactosyltransferase and an appropriate galactose donor.The molecule is then contacted with sialyltransferase and an appropriatesialic acid donor that has been conjugated via a linker tomannose-6-phosphate. In FIG. 56E, alpha-galactosidase A expressed in andsecreted from various mammalian and insect cell systems is remodeled bythe addition of one or more terminal sialic acid-PEG moieties. Thealpha-galactosidase A molecule is contacted with sialidase to removeterminal sialic acid moieties. The molecule is then contacted withsialyltransferase and an appropriate sialic acid donor that has beenderivatized with a PEG moiety. In FIG. 56F, alpha-galactosidase Aexpressed in mammalian, insect, yeast or fungi systems, is remodeled bythe addition of one or more terminal mannose-linker-ApoE moieties. Thealpha-galactosidase A molecule is contacted with mannosyltransferase andan appropriate mannose donor that has been conjugated via a linker toApoE. In FIG. 56G, alpha-galactosidase A expressed in mammalian, insect,yeast or fungal systems is remodeled by the addition ofgalactose-linker-alpha2-macroglobulin moieties. The alpha-galactosidaseA molecule is contacted with Endo-H to trim back glycosyl groups. Themolecule is then contacted with galactosyltransferase and an appropriategalactose donor that has been conjugated via a linker toalpha2-macroglobulin. In FIG. 56H, alpha-galactosidase A expressed ininsect, yeast and fungal systems, is remodeled by the addition of one ormore N-acetylglucosamine-PEG-mannose-6-phosphate moieties. Thealpha-galactosidase molecule is contacted with GnT-I and an appropriateN-acetyl-glucosamine donor that has been derivatized with PEG andmannose-6-phosphate. In FIG. 56I, alpha-galactosidase A expressed ininsect, yeast or fungal systems, is remodeled by the addition of one ormore terminal galactose-PEG-transferrin moieties. Thealpha-galactosidase A molecule is contacted with GnT-I and anappropriate N-acetyl-glucosamine donor. The molecule is then contactedwith galactosyltransferase and an appropriate galactose donor that hasbeen derivatized with PEG and transferrin. In FIG. 56J,alpha-galactosidase A expressed in insect, yeast or fungi systems isremodeled by the addition of one or more terminal sialicacid-PEG-melanotransferrin moieties. The alpha-galactosidase A moleculeis contacted with GnT-I and GnT-II and an appropriateN-acetyl-glucosamine donor. The molecule is then contacted withgalactosyltransferase and an appropriate galactose donor. The moleculeis then contacted with sialyltransferase and an appropriate sialic aciddonor that has been derivatized with PEG and melanotransferrin.

[0914] In another exemplary embodiment, the invention provides methodsfor modifying alpha-iduronidase (Aldurazyme™). FIGS. 57A to 57J presentsome examples. In FIG. 57B, alpha-iduronidase expressed in and secretedfrom various mammalian and insect systems is remodeled by the additionof one or more terminal galactose-PEG-transferrin moieties. Thealpha-iduronidase molecule is contacted with Endo-H to trim backglycosyl groups. The molecule is then contacted withgalactosyltransferase and an appropriate galactose donor that has beenderivatized with PEG and transferrin. In FIG. 57C, alpha-iduronidaseexpressed in and secreted from various mammal and insect cell systems isremodeled by the addition of terminal sialicacid-linker-mannose-6-phosphate moieties. The alpha-iduronidase moleculeis contacted with sialidase to remove terminal sialic acid moieties. Themolecule is then contacted with ST3Gal3 and an appropriate sialic aciddonor that has been conjugated via a linker to mannose-6-phosphate. InFIG. 57D, alpha-iduronidase expressed in NSO murine myeloma cells isremodeled by the addition of one or more terminal sialicacid-linker-mannose-6-phosphate moieties. The alpha-iduronidase moleculeis contacted with sialidase and α-galactosidase to remove terminalsialic acid and galactose moieties. The molecule is then contacted withgalactosyltransferase and an appropriate galactose donor. The moleculeis further contacted with sialyltransferase and an appropriate sialicacid donor that has been conjugated via a linker to mannose-6-phosphate.In FIG. 57E, alpha-iduronidase expressed in and secreted from variousmammalian and insect cell systems is remodeled by the addition of one ormore terminal sialic acid-PEG moieties. The alpha-iduronidase moleculeis contacted with sialidase to remove terminal sialic acid moieties. Themolecule is further contacted with sialyltransferase and an appropriatesialic acid donor that has been derivatized with a PEG moiety. In FIG.57F, alpha-iduronidase expressed in mammalian, insect, yeast or fungisystems is remodeled by the addition of one or more terminalmannose-linker-ApoE moieties. The alpha-iduronidase molecule iscontacted with mannosyltransferase and an appropriate mannose donor thathas been conjugated via a linker to ApoE. In FIG. 57G, alpha-iduronidaseexpressed in mammalian, insect, yeast or fungal systems is remodeled bythe addition of one or more galactose-linker-alpha2-macroglobulinmoieties. The alpha-iduronidase molecule is contacted with Endo-H totrim back glycosyl groups. The molecule is then contacted withgalactosyltransferase and an appropriate galactose donor that has beenconjugated via a linker to alpha2-macroglobulin. In FIG. 57H,alpha-iduronidase expressed in insect, yeast and fungal systems, isremodeled by the addition of one or moreN-acetylglucosamine-PEG-mannose-6-phosphate moieties. Thealpha-galactosidase molecule is contacted with GnT-I and an appropriateN-acetyl-glucosamine donor that has been derivatized with PEG andmannose-6-phosphate. In FIG. 57I, alpha-iduronidase expressed in insect,yeast or fungal systems, is remodeled by the addition of one or moreterminal galactose-PEG-transferrin moieties. The alpha-iduronidasemolecule is contacted with GnT-I and an appropriate N-acetyl-glucosaminedonor. The molecule is then contacted with galactosyltransferase and anappropriate galactose donor that has been derivatized with PEG andtransferrin. In FIG. 57J, alpha-iduronidase expressed in insect, yeastor fungi systems, is remodeled by the addition of one or more terminalsialic acid-PEG-melanotransferrin moieties. The alpha-iduronidasemolecule is contacted with GnT-I and GnT-II and an appropriateN-acetyl-glucosamine donor. The molecule is then contacted withgalactosyltransferase and an appropriate galactose donor. The moleculeis further contacted with sialyltransferase and an appropriate sialicacid donor that has been derivatized with PEG and melanotransferrin.

[0915] A. Creation or Elimination of N-Linked Glycosylation Sites

[0916] The present invention contemplates the use of peptides in whichthe site of the glycan chain(s) on the peptide have been altered fromthat of the native peptide. Typically, N-linked glycan chains are linkedto the primary peptide structure at asparagine residues where theasparagine residue is within an amino acid sequence that is recognizedby a membrane-bound glycosyltransferase in the endoplasmic reticulum(ER). Typically, the recognition site on the primary peptide structureis the sequence asparagine-X-serine/threonine where X can be any aminoacid except proline and aspartic acid. While this recognition site istypical, the invention further encompasses peptides that have N-linkedglycan chains at other recognition sites where the N-linked chains areadded using natural or recombinant glycosyltransferases.

[0917] Since the recognition site for N-linked glycosylation of apeptide is known, it is within the skill of persons in the art to createmutated primary peptide sequences wherein a native N-linkedglycosylation recognition site is removed, or alternatively or inaddition, one or more additional N-glycosylation recognition sites arecreated. Most simply, an asparagine residue can be removed from theprimary sequence of the peptide thereby removing the attachment site fora glycan, thus removing one glycan from the mature peptide. For example,a native recognition site with the sequence of asparagine-serine-serinecan be genetically engineered to have the sequenceleucine-serine-serine, thus eliminating a N-linked glycosylation site atthis position.

[0918] Further, an N-linked glycosylation site can be removed byaltering the residues in the recognition site so that even though theasparagine residue is present, one or more of the additional recognitionresidues are absent. For example, a native sequence ofasparagine-serine-serine can be mutated to asparagine-serine-lysine,thus eliminating an N-glycosylation site at that position. In the caseof N-linked glycosylation sites comprising residues other than thetypical recognition sites described above, the skilled artisan candetermine the sequence and residues required for recognition by theappropriate glycosyltransferase, and then mutate at least one residue sothe appropriate glycosyltransferase no longer recognizes that site. Inother words, it is well within the skill of the artisan to manipulatethe primary sequence of a peptide such that glycosylation sites areeither created or are removed, or both, thereby generating a peptidehaving an altered glycosylation pattern. The invention should thereforenot be construed to be limited to any primary peptide sequence providedherein as the sole sequence for glycan remodeling, but rather should beconstrued to include any and all peptide sequences suitable for glycanremodeling.

[0919] To create a mutant peptide, the nucleic acid sequence encodingthe primary sequence of the peptide is altered so that native codonsencoding native amino acid residues are mutated to generate a codonencoding another amino acid residue. Techniques for altering nucleicacid sequence are common in the art and are described for example in anywell-known molecular biology manual.

[0920] In addition, the nucleic acid encoding a primary peptidestructure can be synthesized in vitro, using standard techniques. Forexample, a nucleic acid molecule can be synthesized in a “gene machine”using protocols such as the phosphoramidite method. Ifchemically-synthesized double stranded DNA is required for anapplication such as the synthesis of a nucleic acid or a fragmentthereof, then each complementary strand is synthesized separately. Theproduction of short nucleic acids (60 to 80 base pairs) is technicallystraightforward and can be accomplished by synthesizing thecomplementary strands and then annealing them. For the production oflonger nucleic acids (>300 base pairs), special strategies may berequired, because the coupling efficiency of each cycle during chemicalDNA synthesis is seldom 100%. To overcome this problem, synthetic genes(double-stranded) are assembled in modular form from single-strandedfragments that are from 20 to 100 nucleotides in length. For reviews onpolynucleotide synthesis, see, for example, Glick and Pasternak(Molecular Biotechnology, Principles and Applications of RecombinantDNA, 1994, ASM Press), Itakura et al. (1984, Annu. Rev. Biochem.53:323), and Climie et al. (1990, Proc. Nat'l Acad. Sci. USA 87:633).

[0921] Additionally, changes in the nucleic acid sequence encoding thepeptide can be made by site-directed mutagenesis. As will beappreciated, this technique typically employs a phage vector whichexists in both a single stranded and double stranded form. Typicalvectors useful in site-directed mutagenesis include vectors such as theM13 phage. These phage are readily available and their use is generallywell known to those skilled in the art. Double stranded plasmids arealso routinely employed in site-directed mutagenesis which eliminatesthe step of transferring the nucleic acid of interest from a plasmid toa phage.

[0922] In general, site-directed mutagenesis is performed by firstobtaining a single-stranded vector or melting the two strands of adouble stranded vector which includes within its sequence a DNA sequencewhich encodes the desired peptide. An oligonucleotide primer bearing thedesired mutated sequence is prepared generally synthetically. Thisprimer is then annealed with the single-stranded vector, and subjectedto DNA polymerizing enzymes such as E. coli polymerase I Klenowfragment, in order to complete the synthesis of the mutation-bearingstrand. Thus, a heteroduplex is formed wherein one strand encodes theoriginal non-mutated sequence and the second strand bears the desiredmutation. This heteroduplex vector is then used to transform ortransfect appropriate cells, such as E. coli cells, and clones areselected which include recombinant vectors bearing the mutated sequencearrangement. A genetic selection scheme was devised by Kunkel et al.(1987, Kunkel et al., Methods Enzymol. 154:367-382) to enrich for clonesincorporating the mutagenic oligonucleotide. Alternatively, the use ofPCR™ with commercially available thermostable enzymes such as Taqpolymerase may be used to incorporate a mutagenic oligonucleotide primerinto an amplified DNA fragment that can then be cloned into anappropriate cloning or expression vector. The PCR™-mediated mutagenesisprocedures of Tomic et al. (1990, Nucl. Acids Res., 12:1656) and Upenderet al. (1995, Biotechniques, 18:29-31) provide two examples of suchprotocols. A PCR™ employing a thermostable ligase in addition to athermostable polymerase may also be used to incorporate a phosphorylatedmutagenic oligonucleotide into an amplified DNA fragment that may thenbe cloned into an appropriate cloning or expression vector. Themutagenesis procedure described by Michael (1994, Biotechniques16:410-412) provides an example of one such protocol.

[0923] Not all Asn-X-Ser/Thr sequences are N-glycosylated suggesting thecontext in which the motif is presented is important. In anotherapproach, libraries of mutant peptides having novel N-linked consensussites are created in order to identify novel N-linked sites that areglycosylated in vivo and are beneficial to the activity, stability orother characteristics of the peptide.

[0924] As noted previously, the consensus sequence for the addition ofN-linked glycan chains in glycoproteins is Asn-X-Ser/Thr where X can beany amino acid. The nucleotide sequence encoding the amino acid twopositions to the carboxyl terminal side of the Asn may be mutated toencode a Ser and/or Thr residue using standard procedures known to thoseof ordinary skill in the art. As stated above not all Asn-X-Ser/Thrsites are modified by the addition of glycans. Therefore, eachrecombinant mutated glycoprotein must be expressed in a fungal, yeast oranimal or mammalian expression system and analyzed for the addition ofan N-linked glycan chain. The techniques for the characterization ofglycosylation sites are well known to one skilled in the art. Further,the biological function of the mutated recombinant glycoprotein can bedetermined using assays standard for the particular protein beingexamined. Thus, it becomes a simple matter to manipulate the primarysequence of a peptide and identify novel glycosylation sites containedtherein, and further determine the effect of the novel site on thebiological activity of the peptide.

[0925] In an alternative embodiment, the nucleotide sequence encodingthe amino acid two positions to the amino terminal side of Ser/Thrresidues may be mutated to encode an Asn using standard procedures knownto those of ordinary skill in the art. The procedures to determinewhether a novel glycosylation site has been created and the effect ofthis site on the biological activity of the peptide are described above.

[0926] B. Creation or elimination of O-linked glycosylation sites Theaddition of an O-linked glycosylation site to a peptide is convenientlyaccomplished by altering the primary amino acid sequence of the peptidesuch that it contains one or more additional O-linked glycosylationsites compared with the beginning primary amino acid sequence of thepeptide. The addition of an O-linked glycosylation site to the peptidemay also be accomplished by incorporation of one or more amino acidspecies into the peptide which comprises an —OH group, preferably serineor threonine residues, within the sequence of the peptide, such that theOH group is accessible and available for O-linked glycosylation. Similarto the discussion of alteration of N-linked glycosylation sites in apeptide, the primary amino acid sequence of the peptide is preferablyaltered at the nucleotide level. Specific nucleotides in the DNAsequence encoding the peptide may be altered such that a desired aminoacid is encoded by the sequence. Mutation(s) in DNA are preferably madeusing methods known in the art, such as the techniques ofphosphoramidite method DNA synthesis and site-directed mutagenesisdescribed above.

[0927] Alternatively, the nucleotide sequence encoding a putative sitefor O-linked glycan addition can be added to the DNA molecule in one orseveral copies to either 5′ or the 3′ end of the molecule. The alteredDNA sequence is then expressed in any one of a fungal, yeast, or animalor mammalian expression system and analyzed for the addition of thesequence to the peptide and whether or not this sequence is a functionalO-linked glycosylation site. Briefly, a synthetic peptide acceptorsequence is introduced at either the 5′ or 3′ end of the nucleotidemolecule. In principle, the addition of this type of sequence is lessdisruptive to the resulting glycoprotein when expressed in a suitableexpression system. The altered DNA is then expressed in CHO cells orother suitable expression system and the proteins expressed thereby areexamined for the presence of an O-linked glycosylation site. Inaddition, the presence or absence of glycan chains can be determined.

[0928] In yet another approach, advantageous sites for new O-linkedsites may be found in a peptide by creating libraries of the peptidecontaining various new O-linked sites. For example, the consensus aminoacid sequence for N-acetylgalactosamine addition by anN-acetylgalactosaminyltransferase depends on the specific transferaseused. The amino acid sequence of a peptide may be scanned to identifycontiguous groups of amino acids that can be mutated to generatepotential sites for addition of O-linked glycan chains. These mutationscan be generated using standard procedures known to those of ordinaryskill in the art as described previously. In order to determine if anydiscovered glycosylation site is actually glycosylated, each recombinantmutated peptide is then expressed in a suitable expression system and issubsequently analyzed for the addition of the site and/or the presenceof an O-linked glycan chain.

[0929] C. Chemical Synthesis of Peptides

[0930] While the primary structure of peptides useful in the inventioncan be generated most efficiently in a cell-based expression system, itis within the scope of the present invention that the peptides may begenerated synthetically. Chemical synthesis of peptides is well known inthe art and include, without limitation, stepwise solid phase synthesis,and fragment condensation either in solution or on solid phase. Aclassic stepwise solid phase synthesis of involves covalently linking anamino acid corresponding to the carboxy-terminal amino acid of thedesired peptide chain to a solid support and extending the peptide chaintoward the amino end by stepwise coupling of activated amino acidderivatives having activated carboxyl groups. After completion of theassembly of the fully protected solid phase bound peptide chain, thepeptide-solid phase covalent attachment is cleaved by suitable chemistryand the protecting groups are removed to yield the product peptide. See,R. Merrifield, Solid Phase Peptide Synthesis: The Synthesis of aTetrapeptide, J. Am. Chem. Soc., 85:2149-2154 (1963). The longer thepeptide chain, the more challenging it is to obtain high-puritywell-defined products. Due to the production of complex mixtures, thestepwise solid phase synthesis approach has size limitations. Ingeneral, well-defined peptides of 100 contiguous amino acid residues ormore are not routinely prepared via stepwise solid phase synthesis.

[0931] The segment condensation method involves preparation of severalpeptide segments by the solid phase stepwise method, followed bycleavage from the solid phase and purification of these maximallyprotected segments. The protected segments are condensed one-by-one tothe first segment, which is bound to the solid phase.

[0932] The peptides useful in the present invention may be synthesizedby exclusive solid phase synthesis, partial solid phase methods,fragment condensation or classical solution synthesis. These synthesismethods are well-known to those of skill in the art (see, for example,Merrifield, J. Am. Chem. Soc. 85:2149 (1963), Stewart et al., “SolidPhase Peptide Synthesis” (2nd Edition), (Pierce Chemical Co. 1984),Bayer and Rapp, Chem. Pept. Prot. 3:3 (1986), Atherton et al., SolidPhase Peptide Synthesis: A Practical Approach (IRL Press 1989), Fieldsand Colowick, “Solid-Phase Peptide Synthesis,” Methods in EnzymologyVolume 289 (Academic Press 1997), and Lloyd-Williams et al., ChemicalApproaches to the Synthesis of Peptides and Peptides (CRC Press, Inc.1997)). Variations in total chemical synthesis strategies, such as“native chemical ligation” and “expressed peptide ligation” are alsostandard (see, for example, Dawson et al., Science 266:776 (1994),Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997), Dawson,Methods Enzymol. 287: 34 (1997), Muir et al, Proc. Nat'l Acad. Sci. USA95:6705 (1998), and Severinov and Muir, J. Biol. Chem. 273:16205(1998)). Also useful are the solid phase peptide synthesis methodsdeveloped by Gryphon Sciences, South San Francisco, Calif. See, U.S.Pat. Nos. 6,326,468, 6,217,873, 6,174,530, and 6,001,364, all of whichare incorporated in their entirety by reference herein.

[0933] D. Post-Translational Modifications

[0934] It will be appreciated to one of ordinary skill in the art thatpeptides may undergo post-translational modification besides theaddition of N-linked and/or O-linked glycans thereto. It is contemplatedthat peptides having post-translational modifications other thanglycosylation can be used as peptides in the invention, as long as thedesired biological activity or function of the peptide is maintained orimproved. Such post-translational modifications may be naturalmodifications usually carried out in vivo, or engineered modificationsof the peptide carried out in vitro. Contemplated known modificationsinclude, but are not limited to, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent crosslinks, formation of cysteine, formation of pyroglutamate,formylation, gamma carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto peptides such as arginylation, and ubiquitination. Enzymes that maybe used to carry out many of these modifications are well known in theart, and available commercially from companies such as BoehringerMannheim (Indianapolis, Ind.) and Sigma Chemical Company (St. Louis,Mo.), among others.

[0935] Such modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Peptides—Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Post-translational Covalent Modification of Peptides, B. C. Johnson,Ed., Academic Press, New York 1-12 (1983); Seifter et al. (Meth.Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci.663:48-62 (1992)).

[0936] Covalent modifications of a peptide may also be introduced intothe molecule in vitro by reacting targeted amino-acid residues of thepeptide with an organic derivatizing agent that is capable of reactingwith selected side chains or terminal amino-acid residues. Most commonlyderivatized residues are cysteinyl, histidyl, lysinyl, arginyl, tyrosyl,glutaminyl, asparaginyl and amino terminal residues. Hydroxylation ofproline and lysine, phosphorylation of hydroxyl groups of seryl andthreonyl residues, methylation of the alpha-amino groups of lysine,histidine, and histidine side chains, acetylation of the N-terminalamine and amidation of the C-terminal carboxylic groups. Suchderivatized moieties may improve the solubility, absorption, biologicalhalf life and the like. The moieties may also eliminate or attenuate anyundesirable side effect of the peptide and the like.

[0937] In addition, derivatization with bifunctional agents is usefulfor cross-linking the peptide to water insoluble support matrices or toother macromolecular carriers. Commonly used cross-linking agentsinclude glutaraldehyde, N-hydroxysuccinimide esters, homobifunctionalimidoesters, 1,1-bis(-diazoloacetyl)-2-phenylethane, and bifunctionalmaleimides. Derivatizing agents such asmethyl-3-[9p-azidophenyl)]dithiopropioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287 and 3,691,016 may be employed for peptideimmobilization.

[0938] E. Fusion Peptides/Peptides

[0939] Peptides useful in the present invention may comprise fusionpeptides. Fusion peptides are particularly advantageous where biologicaland/or functional characteristics of two peptides are desired to becombined in one peptide molecule. Such fusion peptides can presentcombinations of biological activity and function that are not found innature to create novel and useful molecules of therapeutic andindustrial applications. Biological activities of interest include, butare not limited to, enzymatic activity, receptor and/or ligand activity,immunogenic motifs, and structural domains.

[0940] Such fusion peptides are well known in the art, and the methodsof creation will be well-known to those in the art. For example, a humanα-interferon—human albumin fusion peptide has been made wherein theresulting peptide has the therapeutic benefits of α-interferon combinedwith the long circulating life of albumin, thereby creating atherapeutic composition that allows reduced dosing frequency andpotentially reduced side effects in patients. See, Albuferon™ from HumanGenome Sciences, Inc. and U.S. Pat. No. 5,766,883. Other fusion peptidesinclude antibody molecules that are described elsewhere herein.

[0941] F. Generation of Smaller “Biologically Active” Molecules

[0942] The peptides used in the invention may be variants of nativepeptides, wherein a fragment of the native peptide is used in place ofthe full length native peptide. In addition, pre-pro-, and pre-peptidesare contemplated. Variant peptides may be smaller in size that thenative peptide, and may comprise one or more domains of a largerpeptide. Selection of specific peptide domains can be advantageous whenthe biological activity of certain domains in the peptide is desired,but the biological activity of other domains in the peptide is notdesired. Also included are truncations of the peptide and internaldeletions which may enhance the desired therapeutic effect of thepeptide. Any such forms of a peptide is contemplated to be useful in thepresent invention provided that the desired biological activity of thepeptide is preserved.

[0943] Shorter versions of peptides may have unique advantages not foundin the native peptide. In the case of human albumin, it has been foundthat a truncated form comprising as little as 63% of the native albuminpeptide is advantageous as a plasma volume expander. The truncatedalbumin peptide is considered to be better than the native peptide forthis therapeutic purpose because an individual peptide dose of onlyone-half to two-thirds that of natural-human serum albumin, orrecombinant human serum albumin is required for the equivalent colloidosmotic effect. See U.S. Pat. No. 5,380,712, the entirety of which isincorporated by reference herein.

[0944] Smaller “biologically active” peptides have also been found tohave enhanced therapeutic activity as compared to the native peptide.The therapeutic potential of IL-2 is limited by various side effectsdominated by the vascular leak syndrome. A shorter chemicallysynthesized version of the peptide consisting of residues 1-30corresponding to the entire α-helix was found to fold properly andcontain the natural IL-2 biological activity with out the attending sideeffects.

[0945] G. Generation of Novel Peptides

[0946] The peptide of the invention may be derived from a primarysequence of a native peptide, or may be engineered using any of the manymeans known to those of skill in the art. Such engineered peptides canbe designed and/or selected because of enhanced or novel properties ascompared with the native peptide. For example, peptides may beengineered to have increased enzyme reaction rates, increased ordecreased binding affinity to a substrate or ligand, increased ordecreased binding affinity to a receptor, altered specificity for asubstrate, ligand, receptor or other binding partner, increased ordecreased stability in vitro and/or in vivo, or increased or decreasedimmunogenicity in an animal.

[0947] H. Mutations

[0948] 1. Rational Design Mutation

[0949] The peptides useful in the methods of the invention may bemutated to enhance a desired biological activity or function, todiminish an undesirable property of the peptide, and/or to add novelactivities or functions to the peptide. “Rational peptide design” may beused to generate such altered peptides. Once the amino acid sequence andstructure of the peptide is known and a desired mutation planned, themutations can be made most conveniently to the corresponding nucleicacid codon which encodes the amino acid residue that is desired to bemutated. One of skill in the art can easily determine how the nucleicacid sequence should be altered based on the universal genetic code, andknowledge of codon preferences in the expression system of choice. Amutation in a codon may be made to change the amino acid residue thatwill be polymerized into the peptide during translation. Alternatively,a codon may be mutated so that the corresponding encoded amino acidresidue is the same, but the codon choice is better suited to thedesired peptide expression system. For example, cys-residues may bereplaced with other amino acids to remove disulfide bonds from themature peptide, catalytic domains may be mutated to alter biologicalactivity, and in general, isoforms of the peptide can be engineered.Such mutations can be point mutations, deletions, insertions andtruncations, among others.

[0950] Techniques to mutate specific amino acids in a peptide are wellknown in the art. The technique of site-directed mutagenesis, discussedabove, is well suited for the directed mutation of codons. Theoligonucleotide-mediated mutagenesis method is also discussed in detailin Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York, starting at page 15.51). Systematicdeletions, insertions and truncations can be made using linker insertionmutagenesis, digestion with nuclease Bal31, and linker-scanningmutagenesis, among other method well known to those in the art (Sambrooket al., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York).

[0951] Rational peptide design has been successfully used to increasethe stability of enzymes with respect to thermoinactivation andoxidation. For example, the stability of an enzyme was improved byremoval of asparagine residues in α-amylase (Declerck et al., 2000, J.Mol. Biol. 301:1041-1057), the introduction of more rigid structuralelements such as proline into α-amylase (Igarashi et al., 1999, Biosci.Biotechnol. Biochem. 63:1535-1540) and D-xylose isomerase (Zhu et al.,1999, Peptide Eng. 12:635-638). Further, the introduction of additionalhydrophobic contacts stabilized 3-isopropylmalate dehydrogenase (Akanumaet al., 1999, Eur. J. Biochem. 260:499-504) and formate dehydrogenaseobtained from Pseudomonas sp. (Rojkova et al., 1999, FEBS Lett.445:183-188). The mechanisms behind the stabilizing effect of thesemutations is generally applicable to many peptides. These and similarmutations are contemplated to be useful with respect to the peptidesremodeled in the methods of the present invention.

[0952] 2. Random Mutagenesis Techniques

[0953] Novel peptides useful in the methods of the invention may begenerated using techniques that introduce random mutations in the codingsequence of the nucleic acid. The nucleic acid is then expressed in adesired expression system, and the resulting peptide is assessed forproperties of interest. Techniques to introduce random mutations intoDNA sequences are well known in the art, and include PCR mutagenesis,saturation mutagenesis, and degenerate oligonucleotide approaches. SeeSambrook and Russell (2001, Molecular Cloning, A Laboratory Approach,Cold Spring Harbor Press, Cold Spring Harbor, N.Y.) and Ausubel et al.(2002, Current Protocols in Molecular Biology, John Wiley & Sons, NY).

[0954] In PCR mutagenesis, reduced Taq polymerase fidelity is used tointroduce random mutations into a cloned fragment of DNA (Leung et al.,1989, Technique 1:11-15). This is a very powerful and relatively rapidmethod of introducing random mutations into a DNA sequence. The DNAregion to be mutagenized is amplified using the polymerase chainreaction (PCR) under conditions that reduce the fidelity of DNAsynthesis by Taq DNA polymerase, e.g., by using an altered dGTP/dATPratio and by adding Mn²⁺ to the PCR reaction. The pool of amplified DNAfragments are inserted into appropriate cloning vectors to providerandom mutant libraries.

[0955] Saturation mutagenesis allows for the rapid introduction of alarge number of single base substitutions into cloned DNA fragments(Mayers et al., 1985, Science 229:242). This technique includesgeneration of mutations, e.g., by chemical treatment or irradiation ofsingle-stranded DNA in vitro, and synthesis of a complementary DNAstrand. The mutation frequency can be modulated by modulating theseverity of the treatment, and essentially all possible basesubstitutions can be obtained. Because this procedure does not involve agenetic selection for mutant fragments, both neutral substitutions aswell as those that alter function, are obtained. The distribution ofpoint mutations is not biased toward conserved sequence elements.

[0956] A library of nucleic acid homologs can also be generated from aset of degenerate oligonucleotide sequences. Chemical synthesis of adegenerate oligonucleotide sequences can be carried out in an automaticDNA synthesizer, and the synthetic genes may then be ligated into anappropriate expression vector. The synthesis of degenerateoligonucleotides is known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rdCleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam: Elsevierpp. 273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakuraet al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res.11:477. Such techniques have been employed in the directed evolution ofother peptides (see, for example, Scott et al. (1990) Science249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al.(1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; aswell as U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

[0957] a. Directed Evolution

[0958] Peptides useful in the methods of the invention may also begenerated using “directed evolution” techniques. In contrast to sitedirected mutagenesis techniques where knowledge of the structure of thepeptide is required, there now exist strategies to generate libraries ofmutations from which to obtain peptides with improved properties withoutknowledge of the structural features of the peptide. These strategiesare generally known as “directed evolution” technologies and aredifferent from traditional random mutagenesis procedures in that theyinvolve subjecting the nucleic acid sequence encoding the peptide ofinterest to recursive rounds of mutation, screening and amplification.

[0959] In some “directed evolution” techniques, the diversity in thenucleic acids obtained is generated by mutation methods that randomlycreate point mutations in the nucleic acid sequence. The point mutationtechniques include, but are not limited to, “error-prone PCR™” (Caldwelland Joyce, 1994; PCR Methods Appl. 2: 28-33; and Ke and Madison, 1997,Nucleic Acids Res. 25: 3371-3372), repeated oligonucleotide-directedmutagenesis (Reidhaar-Olson et al., 1991, Methods Enzymol. 208:564-586),and any of the aforementioned methods of random mutagenesis.

[0960] Another method of creating diversity upon which directedevolution can act is the use of mutator genes. The nucleic acid ofinterest is cultured in a mutator cell strain the genome of whichtypically encodes defective DNA repair genes (U.S. Pat. No. 6,365,410;Selifonova et al., 2001, Appl. Environ. Microbiol. 67:3645-3649;Long-McGie et al., 2000, Biotech. Bioeng. 68:121-125; see, GenencorInternational Inc, Palo Alto Calif.).

[0961] Achieving diversity using directed evolution techniques may alsobe accomplished using saturation mutagenesis along with degenerateprimers (Gene Site Saturation Mutagenesis™, Diversa Corp., San Diego,Calif.). In this type of saturation mutagenesis, degenerate primersdesigned to cover the length of the nucleic acid sequence to bediversified are used to prime the polymerase in PCR reactions. In thismanner, each codon of a coding sequence for an amino acid may be mutatedto encode each of the remaining common nineteen amino acids. Thistechnique may also be used to introduce mutations, deletions andinsertions to specific regions of a nucleic acid coding sequence whileleaving the rest of the nucleic acid molecule untouched. Procedures forthe gene saturation technique are well known in the art, and can befound in U.S. Pat. No. 6,171,820.

[0962] b. DNA Shuffling

[0963] Novel peptides useful in the methods of the invention may also begenerated using the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”). DNA shuffling techniques are may be employed to modulatethe activities of peptides useful in the invention and may be used togenerate peptides having altered activity. See, generally, U.S. Pat.Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, andStemmer et al. (1994, Nature 370(6488):389-391); Crameri et al. (1998,Nature 391 (6664):288-291); Zhang et al. (1997, Proc. Natl. Acad. Sci.USA 94(9):4504-4509); Stemmer et al. (1994, Proc. Natl. Acad. Sci USA91(22):10747-10751), Patten et al. (1997, Curr. Opinion Biotechnol.8:724-33); Harayama, (1998, Trends Biotechnol. 16(2):76-82); Hansson, etal., (1999, J. Mol. Biol. 287:265-76); and Lorenzo and Blasco (1998,Biotechniques 24(2):308-13) (each of these patents are herebyincorporated by reference in its entirety).

[0964] DNA shuffling involves the assembly of two or more DNA segmentsby homologous or site-specific recombination to generate variation inthe polynucleotide sequence. DNA shuffling has been used to generatenovel variations of human immunodeficiency virus type 1 proteins (Pekrunet al., 2002, J. Virol. 76(6):2924-35), triazine hydrolases (Raillard etal. 2001, Chem Biol 8(9):891-898), murine leukemia virus (MLV) proteins(Powell et al. 2000, Nat Biotechnol 18(12):1279-1282), andindoleglycerol phosphate synthase (Merz et al. 2000, Biochemistry39(5):880-889).

[0965] The technique of DNA shuffling was developed to generatebiomolecular diversity by mimicking natural recombination by allowing invitro homologous recombination of DNA (Stemmler, 1994, Nature 370:389-391; and Stemmler, 1994, PNAS 91: 10747-10751). Generally, in thismethod a population of related genes is fragmented and subjected torecursive cycles of denaturation, rehybridization, followed by theextension of the 5′ overhangs by Taq polymerase. With each cycle, thelength of the fragments increases, and DNA recombination occurs whenfragments originating from different genes hybridize to each other. Theinitial fragmentation of the DNA is usually accomplished by nucleasedigestion, typically using DNase (see Stemmler references, above), butmay also be accomplished by interrupted PCR synthesis (U.S. Pat. No.5,965,408, incorporated herein by reference in its entirety; see,Diversa Corp., San Diego, Calif.). DNA shuffling methods have advantagesover random point mutation methods in that direct recombination ofbeneficial mutations generated by each round of shuffling is achievedand there is therefore a self selection for improved phenotypes ofpeptides.

[0966] The techniques of DNA shuffling are well known to those in art.Detailed explanations of such technology is found in Stemmler, 1994,Nature 370: 389-391 and Stemmler, 1994, PNAS 91: 10747-10751. The DNAshuffling technique is also described in U.S. Pat. Nos. 6,180,406,6,165,793, 6,132,970, 6,117,679, 6,096,548, 5,837,458, 5,834,252,5,830,721, 5,811,238, and 5,605,793 (all of which are incorporated byreference herein in their entirety).

[0967] The art also provides even more recent modifications of the basictechnique of DNA shuffling. In one example, exon shuffling, exons orcombinations of exons that encode specific domains of peptides areamplified using chimeric oligonucleotides. The amplified molecules arethen recombined by self-priming PCR assembly (Kolkman and Stemmler,2001, Nat. Biotech. 19:423-428). In another example, using the techniqueof random chimeragenesis on transient templates (RACHITT) libraryconstruction, single stranded parental DNA fragments are annealed onto afull-length single-stranded template (Coco et al., 2001, Nat.Biotechnol. 19:354-359). In yet another example, staggered extensionprocess (StEP), thermocycling with very abbreviated annealing/extensioncycles is employed to repeatedly interrupt DNA polymerization fromflanking primers (Zhao et al., 1998, Nat. Biotechnol. 16: 258-261). Inthe technique known as CLERY, in vitro family shuffling is combined within vivo homologous recombination in yeast (Abecassis et al., 2000,Nucleic Acids Res. 28:E88;). To maximize intergenic recombination,single stranded DNA from complementary strands of each of the nucleicacids are digested with DNase and annealed (Kikuchi et al., 2000, Gene243:133-137). The blunt ends of two truncated nucleic acids of variablelengths that are linked by a cleavable sequence are then ligated togenerate gene fusion without homologous recombination (Sieber et al.,2001, Nat Biotechnol. 19:456-460; Lutz et al., 2001, Nucleic Acids Res.29:E16; Ostermeier et al., 1999, Nat. Biotechnol. 17:1205-1209; Lutz andBenkovic, 2000, Curr. Opin. Biotechnol. 11:319-324). Recombinationbetween nucleic acids with little sequence homology in common has alsobeen enhanced using exonuclease-mediated blunt-ending of DNA fragmentsand ligating the fragments together to recombine them (U.S. Pat. No.6,361,974, incorporated herein by reference in its entirety). Theinvention contemplates the use of each and every variation describedabove as a means of enhancing the biological properties of any of thepeptides and/or enzymes useful in the methods of the invention.

[0968] In addition to published protocols detailing directed evolutionand gene shuffling techniques, commercial services are now availablethat will undertake the gene shuffling and selection procedures onpeptides of choice. Maxygen (Redwood City, Calif.) offers commercialservices to generate custom DNA shuffled libraries. In addition, thiscompany will perform customized directed evolution procedures includinggene shuffling and selection on a peptide family of choice.

[0969] Optigenix, Inc. (Newark, Del.) offers the related service ofplasmid shuffling. Optigenix uses families of genes to obtain mutantstherein having new properties. The nucleic acid of interest is clonedinto a plasmid in an Aspergillus expression system. The DNA of therelated family is then introduced into the expression system andrecombination in conserved regions of the family occurs in the host.Resulting mutant DNAs are then expressed and the peptide producedtherefrom are screened for the presence of desired properties and theabsence of undesired properties.

[0970] c. Screening Procedures

[0971] Following each recursive round of “evolution,” the desiredpeptides expressed by mutated genes are screened for characteristics ofinterest. The “candidate” genes are then amplified and pooled for thenext round of DNA shuffling. The screening procedure used is highlydependant on the peptide that is being “evolved” and the characteristicof interest. Characteristics such as peptide stability, biologicalactivity, antigenicity, among others can be selected using proceduresthat are well known in the art. Individual assays for the biologicalactivity of preferred peptides useful in the methods of the inventionare described elsewhere herein.

[0972] d. Combinations of Techniques

[0973] It will be appreciated by the skilled artisan that the abovetechniques of mutation and selection can be combined with each other andwith additional procedures to generate the best possible peptidemolecule useful in the methods of the invention. Thus, the invention isnot limited to any one method for the generation of peptides, and shouldbe construed to encompass any and all of the methodology describedherein. For example, a procedure for introducing point mutations into anucleic acid sequence may be performed initially, followed by recursiverounds of DNA shuffling, selection and amplification. The initialintroduction of point mutations may be used to introduce diversity intoa gene population where it is lacking, and the following round of DNAshuffling and screening will select and recombine advantageous pointmutations.

[0974] III. Glycosidases and Glycotransferases

[0975] A. Glycosidases

[0976] Glycosidases are glycosyltransferases that use water as anacceptor molecule, and as such, are typically glycoside-hydrolyticenzymes. Glycosidases can be used for the formation of glycosidic bondsin vitro by controlling the thermodynamics or kinetics of the reactionmixture. Even with modified reaction conditions, though, glycosidasereactions can be difficult to work with, and glycosidases tend to givelow synthetic yields as a result of the reversible transglycosylasereaction and the competing hydrolytic reaction.

[0977] A glycosidase can function by retaining the stereochemistry atthe bond being broken during hydrolysis or by inverting thestereochemistry at the bond being broken during hydrolysis, classifyingthe glycosidase as either a “retaining” glycosidase or an “inverting”glycosidase, respectively. Retaining glycosidases have two criticalcarboxylic acid moieties present in the active site, with onecarboxylate acting as an acid/base catalyst and the other as anucleophile, whereas with the inverting glycosidases, one carboxylicacid functions as an acid and the other functions as a base.

[0978] Methods to determine the activity and linkage specificity of anyglycosidase are well known in the art, including a simplified HPLCprotocol (Jacob and Scudder, 1994, Methods in Enzymol. 230: 280-300). Ageneral discussion of glycosidases and glycosidase treatment is found inGlycobiology, A Practical Approach, (1993, Fukuda and Kobata eds.,Oxford University Press Inc., New York).

[0979] Glycosidases useful in the invention include, but are not limitedto, sialidase, galactosidase, endoglycanase, mannosidase (i.e., αand β,ManI, ManII and ManIII,) xylosidase, fucosidase, Agrobacterium sp.β-glucosidase, Cellulomonas fimi mannosidase 2A, Humicola insolensglycosidase, Sulfolobus solfataricus glycosidase and Bacilluslicheniformis glycosidase.

[0980] The choice of fucosidases for use in the invention depends on thelinkage of the fucose to other molecules. The specificities of manyα-fucosidases useful in the methods of the invention are well known tothose in the art, and many varieties of fucosidase are also commerciallyavailable (Glyko, Novato, Calif.; PROzyme, San Leandro, Calif.;Calbiochem-Novabiochem Corp., San Diego, Calif.; among others).α-Fucosidases of interest include, but are not limited to, α-fucosidasesfrom Turbo cornutus, Charonia lampas, Bacillus fulminans, Aspergillusniger, Clostridium perfringens, Bovine kidney (Glyko), chicken liver(Tyagarajan et al., 1996, Glycobiology 6:83-93) and α-fucosidase II fromXanthomonas manihotis (Glyko, PROzyme). Chicken liver fucosidase isparticularly useful for removal of core fucose from N-linked glycans.

[0981] B. Glycosyltransferases

[0982] Glycosyltransferases catalyze the addition of activated sugars(donor NDP-sugars), in a step-wise fashion, to a protein, glycopeptide,lipid or glycolipid or to the non-reducing end of a growingoligosaccharide. N-linked glycopeptides are synthesized via atransferase and a lipid-linked oligosaccharide donor Dol-PP-NAG₂Glc₃Man₉in an en block transfer followed by trimming of the core. In this casethe nature of the “core” saccharide is somewhat different fromsubsequent attachments. A very large number of glycosyltransferases areknown in the art.

[0983] The glycosyltransferase to be used in the present invention maybe any as long as it can utilize the modified sugar as a sugar donor.Examples of such enzymes include Leloir pathway glycosyltransferases,such as galactosyltransferase, N-acetylglucosaminyltransferase,N-acetylgalactosaminyltransferase, fucosyltransferase,sialyltransferase, mannosyltransferase, xylosyltransferase,glucurononyltransferase and the like.

[0984] For enzymatic saccharide syntheses that involveglycosyltransferase reactions, glycosyltransferases can be cloned, orisolated from any source. Many cloned glycosyltransferases are known, asare their polynucleotide sequences. See, e.g., Taniguchi et al., 2002,Handbook of glycosyltransferases and related genes, Springer, Tokyo.

[0985] Glycosyltransferase amino acid sequences and nucleotide sequencesencoding glycosyltransferases from which the amino acid sequences can bededuced are also found in various publicly available databases,including GenBank, Swiss-Prot, EMBL, and others.

[0986] Glycosyltransferases that can be employed in the methods of theinvention include, but are not limited to, galactosyltransferases,fucosyltransferases, glucosyltransferases,N-acetylgalactosaminyltransferases, N-acetylglucosaminyltransferases,glucuronyltransferases, sialyltransferases, mannosyltransferases,glucuronic acid transferases, galacturonic acid transferases, andoligosaccharyltransferases. Suitable glycosyltransferases include thoseobtained from eukaryotes, as well as from prokaryotes.

[0987] DNA encoding glycosyltransferases may be obtained by chemicalsynthesis, by screening reverse transcripts of mRNA from appropriatecells or cell line cultures, by screening genomic libraries fromappropriate cells, or by combinations of these procedures. Screening ofmRNA or genomic DNA may be carried out using oligonucleotide probesgenerated from the glycosyltransferases nucleic acid sequence. Probesmay be labeled with a detectable label, such as, but not limited to, afluorescent group, a radioactive atom or a chemiluminescent group inaccordance with known procedures and used in conventional hybridizationassays. In the alternative, glycosyltransferases nucleic acid sequencesmay be obtained by use of the polymerase chain reaction (PCR) procedure,with the PCR oligonucleotide primers being produced from theglycosyltransferases nucleic acid sequence. See, U.S. Pat. No. 4,683,195to Mullis et al. and U.S. Pat. No. 4,683,202 to Mullis.

[0988] A glycosyltransferases enzyme may be synthesized in a host celltransformed with a vector containing DNA encoding theglycosyltransferases enzyme. A vector is a replicable DNA construct.Vectors are used either to amplify DNA encoding the glycosyltransferasesenzyme and/or to express DNA which encodes the glycosyltransferasesenzyme. An expression vector is a replicable DNA construct in which aDNA sequence encoding the glycosyltransferases enzyme is operably linkedto suitable control sequences capable of effecting the expression of theglycosyltransferases enzyme in a suitable host. The need for suchcontrol sequences will vary depending upon the host selected and thetransformation method chosen. Generally, control sequences include atranscriptional promoter, an optional operator sequence to controltranscription, a sequence encoding suitable mRNA ribosomal bindingsites, and sequences which control the termination of transcription andtranslation. Amplification vectors do not require expression controldomains. All that is needed is the ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants.

[0989] 1. Fucosyltransferases

[0990] In some embodiments, a glycosyltransferase used in the method ofthe invention is a fucosyltransferase. Fucosyltransferases are known tothose of skill in the art. Exemplary fucosyltransferases includeenzymes, which transfer L-fucose from GDP-fucose to a hydroxy positionof an acceptor sugar. Fucosyltransferases that transfer fromnon-nucleotide sugars to an acceptor are also of use in the presentinvention.

[0991] In some embodiments, the acceptor sugar is, for example, theGlcNAc in a Galβ(1→3,4)GlcNAcβ-group in an oligosaccharide glycoside.Suitable fucosyltransferases for this reaction include theGalβ(1→3,4)GlcNAcβ1-α(1→3,4)fucosyltransferase (FTIII E.C. No.2.4.1.65), which was first characterized from human milk (see, Paicic,et al., Carbohydrate Res. 190: 1-11 (1989); Prieels, et al., J. Biol.Chem. 256: 10456-10463 (1981); and Nunez, et al., Can. J. Chem. 59:2086-2095 (1981)) and the Galβ(1→4)GlcNAcβ-αfucosyltransferases (FTIV,FTV, FTVI) which are found in human serum. FTVII (E.C. No. 2.4.1.65), asialyl α(2→3)Galβ(1→3)GlcNAcβ fucosyltransferase, has also beencharacterized. A recombinant form of the Galβ(1→3,4)GlcNAcβ-α(1→3,4)fucosyltransferase has also been characterized (see,Dumas, et al., Bioorg. Med. Letters 1: 425-428 (1991) andKukowska-Latallo, et al., Genes and Development 4: 1288-1303 (1990)).Other exemplary fucosyltransferases include, for example, α1,2fucosyltransferase (E.C. No. 2.4.1.69). Enzymatic fucosylation can becarried out by the methods described in Mollicone, et al., Eur. J.Biochem. 191: 169-176 (1990) or U.S. Pat. No. 5,374,655.

[0992] 2. Galactosyltransferases

[0993] In another group of embodiments, the glycosyltransferase is agalactosyltransferase. Exemplary galactosyltransferases include α(1,3)galactosyltransferases (E.C. No. 2.4.1.151, see, e.g., Dabkowski et al.,Transplant Proc. 25:2921 (1993) and Yamamoto et al. Nature 345: 229-233(1990), bovine (GenBank j 4989, Joziasse et al., J. Biol. Chem. 264:14290-14297 (1989)), murine (GenBank m26925; Larsen et al., Proc. Nat'l.Acad. Sci. USA 86: 8227-8231 (1989)), porcine (GenBank L36152; Strahanet al., Immunogenetics 41: 101-105 (1995)). Another suitable α1,3galactosyltransferase is that which is involved in synthesis of theblood group B antigen (EC 2.4.1.37, Yamamoto et al., J. Biol. Chem. 265:1146-1151 (1990) (human)).

[0994] Also suitable for use in the methods of the invention are β(1,4)galactosyltransferases, which include, for example, EC 2.4.1.90 (LacNAcsynthetase) and EC 2.4.1.22 (lactose synthetase) (bovine (D'Agostaro etal., Eur. J. Biochem. 183: 211-217 (1989)), human (Masri et al.,Biochem. Biophys. Res. Commun. 157: 657-663 (1988)), murine (Nakazawa etal., J. Biochem. 104: 165-168 (1988)), as well as E.C. 2.4.1.38 and theceramide galactosyltransferase (EC 2.4.1.45, Stahl et al., J. Neurosci.Res. 38: 234-242 (1994)). Other suitable galactosyltransferases include,for example, α1,2 galactosyltransferases (from e.g., Schizosaccharomycespombe, Chapell et al., Mol. Biol. Cell 5: 519-528 (1994)). For furthersuitable galactosyltransferases, see Taniguchi et al. (2002, Handbook ofGlycosyltransferases and Related Genes, Springer, Tokyo), Guo et al.(2001, Glycobiology, 11(10):813-820), and Breton et al. (1998, J.Biochem. 123:1000-1009).

[0995] The production of proteins such as the enzyme GalNAc T_(I-XIV)from cloned genes by genetic engineering is well known. See, e.g., U.S.Pat. No. 4,761,371. One method involves collection of sufficientsamples, then the amino acid sequence of the enzyme is determined byN-terminal sequencing. This information is then used to isolate a cDNAclone encoding a full-length (membrane bound) transferase which uponexpression in the insect cell line Sf9 resulted in the synthesis of afully active enzyme. The acceptor specificity of the enzyme is thendetermined using a semiquantitative analysis of the amino acidssurrounding known glycosylation sites in 16 different proteins followedby in vitro glycosylation studies of synthetic peptides. This work hasdemonstrated that certain amino acid residues are overrepresented inglycosylated peptide segments and that residues in specific positionssurrounding glycosylated serine and threonine residues may have a moremarked influence on acceptor efficiency than other amino acid moieties.

[0996] 3. Sialyltransferases

[0997] Sialyltransferases are another type of glycosyltransferase thatis useful in the recombinant cells and reaction mixtures of theinvention. Examples of sialyltransferases that are suitable for use inthe present invention include ST3Gal III (e.g., a rat or human ST3GalIII), ST3Gal IV, ST3Gal I, ST6Gal I, ST3Gal V, ST6Gal 11, ST6GalNAc I,ST6GalNAc II, and ST6GalNAc III (the sialyltransferase nomenclature usedherein is as described in Tsuji et al., Glycobiology 6: v-xiv (1996)).An exemplary α(2,3)sialyltransferase referred to asα(2,3)sialyltransferase (EC 2.4.99.6) transfers sialic acid to thenon-reducing terminal Gal of a Galβ1→3Glc disaccharide or glycoside.See, Van den Eijnden et al., J. Biol. Chem. 256: 3159 (1981), Weinsteinet al., J. Biol. Chem. 257: 13845 (1982) and Wen et al., J. Biol. Chem.267: 21011 (1992). Another exemplary α2,3-sialyltransferase (EC2.4.99.4) transfers sialic acid to the non-reducing terminal Gal of thedisaccharide or glycoside. see, Rearick et al., J. Biol. Chem. 254: 4444(1979) and Gillespie et al., J. Biol. Chem. 267: 21004 (1992). Furtherexemplary enzymes include Gal-β-1,4-GlcNAc α-2,6 sialyltransferase (See,Kurosawa et al. Eur. J. Biochem. 219: 375-381 (1994)).

[0998] Preferably, for glycosylation of carbohydrates of glycopeptidesthe sialyltransferase will be able to transfer sialic acid to thesequence Galβ1,4GlcNAc-, Galβ1,3GlcNAc-, or Galβ1,3GalNAc-, the mostcommon penultimate sequences underlying the terminal sialic acid onfully sialylated carbohydrate structures (see, Table 8).α2,8-Sialyltransferases capable of transfering sialic acid toα2,3Galβ1,4GlcNAc are also useful in the methods of the invention. TABLE8 Sialyltransferases which use the Galβ1, 4GlcNAc sequence as anacceptor substrate Sialyltransferase Source Sequence(s) formed Ref.ST6Gal I Mammalian NeuAcα2, 6Galβ1, 4GlcNAc- 1 ST3Gal III MammalianNeuAcα2, 3Galβ1, 4GlcNAc- 1 NeuAcα2, 3Galβ1, 3GlcNAc- ST3Gal IVMammalian NeuAcα2, 3Galβ1, 4GlcNAc- 1 NeuAcα2, 3Galβ1, 3GlcNAc- ST6GalII Mammalian NeuAcα2, 6Galβ1, 4GlcNAc- ST6Gal II Photobacterium NeuAcα2,6Galβ1, 4GlcNAc- 2 ST3Gal V N. NeuAcα2, 3Galβ1, 4GlcNAc- 3 meningitidesN. gonorrhoeae

[0999] An example of a sialyltransferase that is useful in the claimedmethods is ST3Gal III, which is also referred to asα(2,3)sialyltransferase (EC 2.4.99.6). This enzyme catalyzes thetransfer of sialic acid to the Gal of a Galβ1,3GlcNAc or Galβ1,4GlcNAcglycoside (see, e.g., Wen et al., J. Biol. Chem. 267: 21011 (1992); Vanden Eijnden et al., J. Biol. Chem. 256: 3159 (1991)) and is responsiblefor sialylation of asparagine-linked oligosaccharides in glycopeptides.The sialic acid is linked to a Gal with the formation of an α-linkagebetween the two saccharides. Bonding (linkage) between the saccharidesis between the 2-position of NeuAc and the 3-position of Gal. Thisparticular enzyme can be isolated from rat liver (Weinstein et al., J.Biol. Chem. 257: 13845 (1982)); the human cDNA (Sasaki et al. (1993) J.Biol. Chem. 268: 22782-22787; Kitagawa & Paulson (1994) J. Biol. Chem.269: 1394-1401) and genomic (Kitagawa et al. (1996) J. Biol. Chem. 271:931-938) DNA sequences are known, facilitating production of this enzymeby recombinant expression. In a preferred embodiment, the claimedsialylation methods use a rat ST3Gal III.

[1000] An example of a sialyltransferase that is useful in the claimedmethods is CST-I from Campylobacter (see, for example, U.S. Pat. Nos.6,503,744, 6,096,529, and 6,210,933 and WO99/49051, and published U.S.Pat. Application 2002/2,042,369). This enzyme catalyzes the transfer ofsialic acid to the Gal of a Galβ1,4Glc or Galβ1,3GalNAc. Other exemplarysialyltransferases of use in the present invention include thoseisolated from Campylobacter jejuni, including the α(2,3)sialyltransferase. See, e.g, WO99/49051.

[1001] Other sialyltransferases, including those listed in Table 8, arealso useful in an economic and efficient large-scale process forsialylation of commercially important glycopeptides. As a simple test tofind out the utility of these other enzymes, various amounts of eachenzyme (1-100 mU/mg protein) are reacted with asialo-α₁ AGP (at 1-10mg/ml) to compare the ability of the sialyltransferase of interest tosialylate glycopeptides relative to either bovine ST6Gal I, ST3Gal IIIor both sialyltransferases. Alternatively, other glycopeptides orglycopeptides, or N-linked oligosaccharides enzymatically released fromthe peptide backbone can be used in place of asialo-α₁ AGP for thisevaluation. Sialyltransferases with the ability to sialylate N-linkedoligosaccharides of glycopeptides more efficiently than ST6Gal I areuseful in a practical large-scale process for peptide sialylation (asillustrated for ST3Gal III in this disclosure).

[1002] 4. Other Glycosyltransferases

[1003] One of skill in the art will understand that otherglycosyltransferases can be substituted into similar transferase cyclesas have been described in detail for the sialyltransferase. Inparticular, the glycosyltransferase can also be, for instance,glucosyltransferases, e.g., Alg8 (Stagljov et al., Proc. Natl. Acad.Sci. USA 91: 5977 (1994)) or Alg5 (Heesen et al., Eur. J. Biochem. 224:71 (1994)).

[1004] N-acetylgalactosaminyltransferases are also of use in practicingthe present invention. Suitable N-acetylgalactosaminyltransferasesinclude, but are not limited to, α(1,3)N-acetylgalactosaminyltransferase, β(1,4)N-acetylgalactosaminyltransferases (Nagata et al., J. Biol. Chem. 267:12082-12089 (1992) and Smith et al., J. Biol. Chem. 269: 15162 (1994))and peptide N-acetylgalactosaminyltransferase (Homa et al., J. Biol.Chem. 268: 12609 (1993)). Suitable N-acetylglucosaminyltransferasesinclude GnT-I (2.4.1.101, Hull et al., BBRC 176: 608 (1991)), GnT-II,GnT-III (Ihara et al., J. Biochem. 113: 692 (1993)), GnT-IV, GnT-V(Shoreibah et al., J. Biol. Chem. 268: 15381 (1993)) and GnT-VI,O-linked N-acetylglucosaminyltransferase (Bierhuizen et al., Proc. Natl.Acad. Sci. USA 89: 9326 (1992)), N-acetylglucosamine-1-phosphatetransferase (Rajput et al., Biochem J. 285: 985 (1992), and hyaluronansynthase.

[1005] Mannosyltransferases are of use to transfer modified mannosemoieties. Suitable mannosyltransferases include α(1,2)mannosyltransferase, α(1,3) mannosyltransferase, α(1,6)mannosyltransferase, β(1,4) mannosyltransferase, Dol-P-Man synthase,OChI, and Pmt1 (see, Kornfeld et al., Annu. Rev. Biochem. 54: 631-664(1985)).

[1006] Xylosyltransferases are also useful in the present invention.See, for example, Rodgers, et al., Biochem. J., 288:817-822 (1992); andElbain, et al., U.S. Pat. No., 6,168,937.

[1007] Other suitable glycosyltransferase cycles are described inIchikawa et al., JACS 114: 9283 (1992), Wong et al., J. Org. Chem. 57:4343 (1992), and Ichikawa et al. in CARBOHYDRATES AND CARBOHYDRATEPOLYMERS. Yaltami, ed. (ATL Press, 1993).

[1008] Prokaryotic glycosyltransferases are also useful in practicingthe invention. Such glycosyltransferases include enzymes involved insynthesis of lipooligosaccharides (LOS), which are produced by many gramnegative bacteria. The LOS typically have terminal glycan sequences thatmimic glycoconjugates found on the surface of human epithelial cells orin host secretions (Preston et al., Critical Reviews in Microbiology23(3): 139-180 (1996)). Such enzymes include, but are not limited to,the proteins of the rfa operons of species such as E. coli andSalmonella typhimurium, which include a β1,6 galactosyltransferase and aβ1,3 galactosyltransferase (see, e.g., EMBL Accession Nos. M80599 andM86935 (E. coli); EMBL Accession No. S56361 (S. typhimurium)), aglucosyltransferase (Swiss-Prot Accession No. P25740 (E. coli), anβ1,2-glucosyltransferase (rfaJ)(Swiss-Prot Accession No. P27129 (E.coli) and Swiss-Prot Accession No. P19817 (S. typhimurium)), and anβ1,2-N-acetylglucosaminyltransferase (rfaK)(EMBL Accession No. U00039(E. coli). Other glycosyltransferases for which amino acid sequences areknown include those that are encoded by operons such as rfaB, which havebeen characterized in organisms such as Klebsiella pneumoniae, E. coli,Salmonella typhimurium, Salmonella enterica, Yersinia enterocolitica,Mycobacterium leprosum, and the rh1 operon of Pseudomonas aeruginosa.

[1009] Also suitable for use in the present invention areglycosyltransferases that are involved in producing structurescontaining lacto-N-neotetraose,D-galactosyl-β-1,4-N-acetyl-D-glucosaminyl-β-1,3-D-galactosyl-β-1,4-D-glucose,and the P^(k) blood group trisaccharide sequence,D-galactosyl-α-1,4-D-galactosyl-β-1,4-D-glucose, which have beenidentified in the LOS of the mucosal pathogens Neisseria gonnorhoeae andN. meningitidis (Scholten et al., J. Med. Microbiol. 41: 236-243(1994)). The genes from N. meningitidis and N. gonorrhoeae that encodethe glycosyltransferases involved in the biosynthesis of thesestructures have been identified from N. meningitidis immunotypes L3 andL1 (Jennings et al., Mol. Microbiol. 18: 729-740 (1995)) and the N.gonorrhoeae mutant F62 (Gotshlich, J. Exp. Med. 180: 2181-2190 (1994)).In N. meningitidis, a locus consisting of three genes, lgtA, lgtB and lgE, encodes the glycosyltransferase enzymes required for addition of thelast three of the sugars in the lacto-N-neotetraose chain (Wakarchuk etal., J. Biol. Chem. 271: 19166-73 (1996)). Recently the enzymaticactivity of the lgtB and lgtA gene product was demonstrated, providingthe first direct evidence for their proposed glycosyltransferasefunction (Wakarchuk et al., J. Biol. Chem. 271(45): 28271-276 (1996)).In N. gonorrhoeae, there are two additional genes, lgtD which addsβ-D-GalNAc to the 3 position of the terminal galactose of thelacto-N-neotetraose structure and lgtC which adds a terminal α-D-Gal tothe lactose element of a truncated LOS, thus creating the P^(k) bloodgroup antigen structure (Gotshlich (1994), supra.). In N. meningitidis,a separate immunotype L1 also expresses the P^(k) blood group antigenand has been shown to carry an lgtC gene (Jennings et al., (1995),supra.). Neisseria glycosyltransferases and associated genes are alsodescribed in U.S. Pat. No. 5,545,553 (Gotschlich). Genes forc1,2-fucosyltransferase and α1,3-fucosyltransferase from Helicobacterpylori has also been characterized (Martin et al., J. Biol. Chem. 272:21349-21356 (1997)). Also of use in the present invention are theglycosyltransferases of Campylobacter jejuni (see, Taniguchi et al.,2002, Handbook of glycosyltransferases and related genes, Springer,Tokyo).

[1010] B. Sulfotransferases

[1011] The invention also provides methods for producing peptides thatinclude sulfated molecules, including, for example sulfatedpolysaccharides such as heparin, heparan sulfate, carragenen, andrelated compounds. Suitable sulfotransferases include, for example,chondroitin-6-sulphotransferase (chicken cDNA described by Fukuta etal., J. Biol. Chem. 270: 18575-18580 (1995); GenBank Accession No.D49915), glycosaminoglycan N-acetylglucosamineN-deacetylase/N-sulphotransferase 1 (Dixon et al., Genomics 26: 239-241(1995); UL18918), and glycosaminoglycan N-acetylglucosamineN-deacetylase/N-sulphotransferase 2 (murine cDNA described in Orellanaet al., J. Biol. Chem. 269: 2270-2276 (1994) and Eriksson et al., J.Biol. Chem. 269: 10438-10443 (1994); human cDNA described in GenBankAccession No. U2304).

[1012] C. Cell-Bound Glycosyltransferases

[1013] In another embodiment, the enzymes utilized in the method of theinvention are cell-bound glycosyltransferases. Although many solubleglycosyltransferases are known (see, for example, U.S. Pat. No.5,032,519), glycosyltransferases are generally in membrane-bound formwhen associated with cells. Many of the membrane-bound enzymes studiedthus far are considered to be intrinsic proteins; that is, they are notreleased from the membranes by sonication and require detergents forsolubilization. Surface glycosyltransferases have been identified on thesurfaces of vertebrate and invertebrate cells, and it has also beenrecognized that these surface transferases maintain catalytic activityunder physiological conditions. However, the more recognized function ofcell surface glycosyltransferases is for intercellular recognition(Roth, 1990, Molecular Approaches to Supracellular Phenomena,).

[1014] Methods have been developed to alter the glycosyltransferasesexpressed by cells. For example, Larsen et al., Proc. Natl. Acad. Sci.USA 86: 8227-8231 (1989), report a genetic approach to isolate clonedcDNA sequences that determine expression of cell surface oligosaccharidestructures and their cognate glycosyltransferases. A cDNA librarygenerated from mRNA isolated from a murine cell line known to expressUDP-galactose:.β.-D-galactosyl-1,4-N-acetyl-D-glucosaminideα-1,3-galactosyltransferase was transfected into COS-1 cells. Thetransfected cells were then cultured and assayed for α 1-3galactosyltransferase activity.

[1015] Francisco et al., Proc. Natl. Acad. Sci. USA 89: 2713-2717(1992), disclose a method of anchoring β-lactamase to the externalsurface of Escherichia coli. A tripartite fusion consisting of (i) asignal sequence of an outer membrane protein, (ii) a membrane-spanningsection of an outer membrane protein, and (iii) a complete matureβ-lactamase sequence is produced resulting in an active surface boundβ-lactamase molecule. However, the Francisco method is limited only toprokaryotic cell systems and as recognized by the authors, requires thecomplete tripartite fusion for proper functioning.

[1016] D. Fusion Enzymes

[1017] In other exemplary embodiments, the methods of the inventionutilize fusion peptides that have more than one enzymatic activity thatis involved in synthesis of a desired glycopeptide conjugate. The fusionpeptides can be composed of, for example, a catalytically active domainof a glycosyltransferase that is joined to a catalytically active domainof an accessory enzyme. The accessory enzyme catalytic domain can, forexample, catalyze a step in the formation of a nucleotide sugar that isa donor for the glycosyltransferase, or catalyze a reaction involved ina glycosyltransferase cycle. For example, a polynucleotide that encodesa glycosyltransferase can be joined, in-frame, to a polynucleotide thatencodes an enzyme involved in nucleotide sugar synthesis. The resultingfusion peptide can then catalyze not only the synthesis of thenucleotide sugar, but also the transfer of the sugar moiety to theacceptor molecule. The fusion peptide can be two or more cycle enzymeslinked into one expressible nucleotide sequence. In other embodimentsthe fusion ppeptide includes the catalytically active domains of two ormore glycosyltransferases. See, for example, U.S. Pat. No. 5,641,668.The modified glycopeptides of the present invention can be readilydesigned and manufactured utilizing various suitable fusion peptides(see, for example, PCT Patent Application PCT/CA98/01180, which waspublished as WO 99/31224 on Jun. 24, 1999.)

[1018] E. Immobilized Enzymes

[1019] In addition to cell-bound enzymes, the present invention alsoprovides for the use of enzymes that are immobilized on a solid and/orsoluble support. In an exemplary embodiment, there is provided aglycosyltransferase that is conjugated to a PEG via an intact glycosyllinker according to the methods of the invention. The PEG-linker-enzymeconjugate is optionally attached to solid support. The use of solidsupported enzymes in the methods of the invention simplifies the work upof the reaction mixture and purification of the reaction product, andalso enables the facile recovery of the enzyme. The glycosyltransferaseconjugate is utilized in the methods of the invention. Othercombinations of enzymes and supports will be apparent to those of skillin the art.

[1020] F. Mutagenesis of Glycosyltransferases

[1021] The novel forms of the glycosyltransferases, sialyltransferases,sulfotransferases, and any other enzymes used in the method of theinvention can be created using any of the methods described previously,as well as others well known to those in the art. Of particular interestare transferases with altered acceptor specificity and/or donorspecificity. Also of interest are enzymes with higher conversion ratesand higher stability among others.

[1022] The techniques of rational design mutagenesis can be used whenthe sequence of the peptide is known. Since the sequences as well asmany of the tertiary structures of the transferases and glucosidasesused in the invention are known, these enzymes are ideal for rationaldesign of mutants. For example, the catalytic site of the enzyme can bemutated to alter the donor and/or acceptor specificity of the enzyme.

[1023] The extensive tertiary structural data on theglycosyltransferases and glycosidase hydrolases also make these enzymeidea for mutations involving domain exchanges. Glycosyltransferases andglycosidase hydrolases are modular enzymes (see, Bourne and Henrissat,2001, Current Opinion in Structural Biology 11:593-600).Glycosyltransferases are divided into two families bases on theirstructure: GT-A and GT-B. The glycosyltransferases of the GT-A familycomprise two dissimilar domains, one involved in nucleotide binding andthe other in acceptor binding. Thus, one could conveniently fuse the DNAsequence encoding the domain from one gene in frame with a domain from asecond gene to create a new gene that encodes a protein with a newacceptor/donor specificity. Such exchanges of domains could additionallyinclude the carbohydrate modules and other accessory domains.

[1024] The techniques of random mutation and/or directed evolution, asdescribed above, may also be used to create novel forms of theglycosyltransferases and glycosidases used in the invention.

[1025] IV. In Vitro and In Vivo Expression Systems

[1026] A. Cells for the Production of Glycopeptides

[1027] The action of glycosyltransferases is key to the glycosylation ofpeptides, thus, the difference in the expression of a set ofglycosyltransferases in any given cell type affects the pattern ofglycosylation on any given peptide produced in that cell. For a reviewof host cell dependent glycosylation of peptides, see Kabata andTakasaki, “Structure and Biosynthesis of Cell Surface Carbohydrates,” inCell Surface Carbohydrates and Cell Development, 1991, pp. 1-24, Eds.Minoru Fukuda, CRC Press, Boca Raton, Fla.

[1028] According to the present disclosure, the type of cell in whichthe peptide is produced is relevant only with respect to the degree ofremodeling required to generate a peptide having desired glycosylation.For example, the number and sequence of enzymatic digestion reactionsand the number and sequence of enzymatic synthetic reactions that arerequired in vitro to generate a peptide having desired glycosylationwill vary depending on the structure of the glycan on the peptideproduced by a particular cell type. While the invention should in no waybe construed to be limited to the production of peptides from any oneparticular cell type including any cell type disclosed herein, adiscussion of several cell systems is now presented which establishesthe power of the present invention and its independence of the cell typein which the peptides are generated.

[1029] In general, and to express a peptide from a nucleic acid encodingit, the nucleic acid must be incorporated into an expression cassette,comprising a promoter element, a terminator element, and the codingsequence of the peptide operably linked between the two. The expressioncassette is then operably linked into a vector. Toward this end,adapters or linkers may be employed to join the nucleotide fragments orother manipulations may be involved to provide for convenientrestriction sites, removal of superfluous nucleotides, removal ofrestriction sites, or the like. For this purpose, in vitro mutagenesis,primer repair, restriction, annealing, resubstitutions, e.g.,transitions and transversions, may be involved. A shuttle vector has thegenetic elements necessary for replication in a cell. Some vectors maybe replicated only in prokaryotes, or may be replicated in bothprokaryotes and eukaryotes. Such a plasmid expression vector will bemaintained in one or more replication systems, preferably tworeplication systems, that allow for stable maintenance within a yeasthost cell for expression purposes, and within a prokaryotic host forcloning purposes. Many vectors with diverse characteristics are nowavailable commercially. Vectors are usually plasmids or phages, but mayalso be cosmids or mini-chromosomes. Conveniently, many commerciallyavailable vectors will have the promoter and terminator of theexpression cassette already present, and a multi-linker site where thecoding sequence for the peptide of interest can be inserted. The shuttlevector containing the expression cassette is then transformed in E. coliwhere it is replicated during cell division to generate a preparation ofvector that is sufficient to transform the host cells of the chosenexpression system. The above methodology is well know to those in theart, and protocols by which to accomplish can be found Sambrook et al.(2001, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York).

[1030] The vector, once purified from the cells in which it isamplified, is then transformed into the cells of the expression system.The protocol for transformation depended on the kind of the cell and thenature of the vector. Transformants are grown in an appropriate nutrientmedium, and, where appropriate, maintained under selective pressure toinsure retention of endogenous DNA. Where expression is inducible,growth can be permitted of the yeast host to yield a high density ofcells, and then expression is induced. The secreted, mature heterologouspeptide can be harvested by any conventional means, and purified bychromatography, electrophoresis, dialysis, solvent-solvent extraction,and the like.

[1031] The techniques of molecular cloning are well-known in the art.Further, techniques for the procedures of molecular cloning can be foundin Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Glover etal., (1985, DNA Cloning: A Practical Approach, Volumes I and II); Gaitet al., (1985, Oligonucleotide Synthesis); Hames and Higgins (1985,Nucleic Acid Hybridization); Hames and Higgins (1984, Transcription AndTranslation); Freshney et al., (1986, Animal Cell Culture); Perbal,(1986, Immobilized Cells And Enzymes, IRL Press); Perbal,(1984, APractical Guide To Molecular Cloning); Ausubel et al. (2002, CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc.).

[1032] B. Fungi and Yeast

[1033] Peptides produced in yeast are glycosylated and the glycanstructures present thereon are primarily high mannose structures. In thecase of N-glycans, the glycan structures produced in yeast may containas many as nine or more mannose residues which may or may not containadditional sugars added thereto. An example of the type of glycan onpeptides produced by yeast cells is shown in FIG. 4, left side.Irrespective of the number of mannose residues and the type andcomplexity of additional sugars added thereto, N-glycans as componentsof peptides produced in yeast cells comprise a trimannosyl corestructure as shown in FIG. 4. When the glycan structure on a peptideproduced by a yeast cell is a high mannose structure, it is a simplematter for the ordinary skilled artisan to remove, in vitro usingavailable mannosidase enzymes, all of the mannose residues from themolecule except for those that comprise the trimannosyl core of theglycan, thereby generating a peptide having an elemental trimannosylcore structure attached thereto. Now, using the techniques available inthe art and armed with the present disclosure, it is a simple matter toenzymatically add, in vitro, additional sugar moieties to the elementaltrimannosyl core structure to generate a peptide having a desired glycanstructure attached thereto. Similarly, when the peptide produced by theyeast cell comprises a high mannose structure in addition to othercomplex sugars attached thereto, it is a simple matter to enzymaticallycleave off all of the additional sugars, including extra mannoseresidues, to arrive at the elemental trimannosyl core structure. Oncethe elemental trimannosyl core structure is produced, generation of apeptide having desired glycosylation is possible following thedirections provided herein.

[1034] By “yeast” is intended ascosporogenous yeasts (Endomycetales),basidiosporogenous yeasts, and yeast belonging to the Fungi Imperfecti(Blastomycetes). The ascosporogenous yeasts are divided into twofamilies, Spermophthoraceae and Saccharomycetaceae. The later iscomprised of four subfamilies, Schizosaccharomycoideae (e.g., genusSchizosaccharomyces), Nadsonioideae, Lipomycoideae, andSaccharomycoideae (e.g., genera Pichia, Kluyveromyces, andSaccharomyces). The basidiosporogenous yeasts include the generaLeucosporidium, Rhodosporidium, Sporidiobolus, Filobasidium, andFilobasidiella. Yeast belonging to the Fungi Imperfecti are divided intotwo families, Sporobolomycetaceae (e.g., genera Sporobolomyces, Bullera)and Cryptococcaceae (e.g., genus Candida). Of particular interest to thepresent invention are species within the genera Saccharomyces, Pichia,Aspergillus, Trichoderna, Kluyveromyces, especially K. lactis and K.drosophilum, Candida, Hansenula, Schizpsaccaromyces, Yarrowia, andChrysoporium. Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Skinner et al., eds. 1980) Biology and Activities of Yeast(Soc. App. Bacteriol. Symp. Series No. 9).

[1035] In addition to the foregoing, those of ordinary skill in the artare presumably familiar with the biology of yeast and the manipulationof yeast genetics. See, for example, Bacila et al., eds. (1978,Biochemistry and Genetics of Yeast, Academic Press, New York); and Roseand Harrison. (1987, The Yeasts (2^(nd) ed.) Academic Press, London).Methods of introducing exogenous DNA into yeast hosts are well known inthe art. There are a wide variety of methods for transformation ofyeast. Spheroplast transformation is taught by Hinnen et al (1978, Proc.Natl. Acad. Sci. USA 75:1919-1933); Beggs, (1978, Nature275(5676):104-109); and Stinchcomb et al., (EPO Publication No. 45,573;herein incorporated by reference), Electroporation is taught by Beckerand Gaurante, (1991, Methods Enzymol. 194:182-187), Lithium acetate istaught by Gietz et al. (2002, Methods Enzymol. 350:87-96) and Mount etal. (1996, Methods Mol. Biol. 53:139-145). For a review oftransformation systems of non-Saccharomyces yeasts, see Wang et al.(Crit Rev Biotechnol. 2001;21(3):177-218). For general procedures onyeast genetic engineering, see Barr et al., (1989, Yeast geneticengineering, Butterworths, Boston).

[1036] In addition to wild-type yeast and fungal cells, there are alsostrains of yeast and fungi that have been mutated and/or selected toenhance the level of expression of the exogenous gene, and the purity,the post-translational processing of the resulting peptide, and therecovery and purity of the mature peptide. Expression of an exogenouspeptide may also be direct to the cell secretory pathway, as illustratedby the expression of insulin (see (Kjeldsen, 2000, Appl. Microbiol.Biotechnol. 54:277-286, and references cited therein). In general, tocause the exogenous peptide to be secreted from the yeast cell,secretion signals derived from yeast genes may be used, such as those ofthe genes of the killer toxin (Stark and Boyd, 1986, EMBO J.5:1995-2002) or of the alpha pheromone (Kurjan and Herskowitz, 1982,Cell 30:933; Brake et al., 1988, Yeast 4:S436).

[1037] Regarding the filamentous fungi in general, methods for geneticmanipulation can be found in Kinghorn and Turner (1992, AppliedMolecular Genetics of Filamentous Fungi, Blackie Academic andProfessional, New York). Guidance on appropriate vectors can be found inMartinelli and Kinghorn (1994, Aspergillus: 50 years, Elsevier,Amsterdam).

[1038] 1. Saccharomyces

[1039] In Saccharomyces, suitable yeast vectors for use producing apeptide include YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76:1035-1039, 1978), YEp13 (Broach et al., Gene 8: 121-133, 1979), POTvectors (Kawasaki et al, U.S. Pat. No. 4,931,373, which is incorporatedby reference herein), pJDB249 and pJDB219 (Beggs, Nature 275:104-108,1978) and derivatives thereof. Preferred promoters for use in yeastinclude promoters for yeast glycolytic gene expression (Hitzeman et al.,J. Biol. Chem. 255: 12073-12080, 1980; Alber and Kawasaki, J. Mol. Appl.Genet. 1: 419-434, 1982; Kawasaki, U.S. Pat. No. 4,599,311) or alcoholdehydrogenase genes (Young et al., in Genetic Engineering ofMicroorganisms for Chemicals, Hollaender et al., (eds.), p. 355, Plenum,New York, 1982; Ammerer, Meth. Enzymol. 101: 192-201, 1983), and theADH2-4C promoter (Russell et al., Nature 304: 652-654, 1983; Irani andKilgore, U.S. patent application Ser. No. 07/784,653, CA 1,304,020 andEP 284 044, which are incorporated herein by reference). The expressionunits may also include a transcriptional terminator. A preferredtranscriptional terminator is the TPI1 terminator (Alber and Kawasaki,ibid.).

[1040] Examples of such yeast-bacteria shuttle vectors include Yep24(Botstein et al. (1979) Gene 8:17-24; pC1 (Brake et al. (1984) Proc.Natl. Acad. Sci. USA 81:4642-4646), and Yrp17 (Stnichomb et al. (1982)J. Mol. Biol. 158:157). Additionally, a plasmid expression vector may bea high or low copy number plasmid, the copy number generally rangingfrom about 1 to about 200. In the case of high copy number yeastvectors, there will generally be at least 10, preferably at least 20,and usually not exceeding about 150 copies of the vector in a singlehost. Depending upon the heterologous peptide selected, either a high orlow copy number vector may be desirable, depending upon the effect ofthe vector and the recombinant peptide on the host. See, for example,Brake et al. (1984) Proc. Natl. Acad. Sci. USA 81:4642-4646. DNAconstructs of the present invention can also be integrated into theyeast genome by an integrating vector. Examples of such vectors areknown in the art. See, for example, Botstein et al. (1979) Gene 8:17-24.

[1041] The selection of suitable yeast and other microorganism hosts forthe practice of the present invention is within the skill of the art. Ofparticular interest are the Saccharomyces species S. cerevisiae, S.carlsbergensis, S. diastaticus, S. douglasii, S. kluyveri, S. norbensis,and S. oviformis. When selecting yeast host cells for expression of adesired peptide, suitable host cells may include those shown to have,inter alia, good secretion capacity, low proteolytic activity, andoverall vigor. Yeast and other microorganisms are generally availablefrom a variety of sources, including the Yeast Genetic Stock Center,Department of Biophysics and Medical Physics, University of California,Berkeley, Calif.; and the American Type Culture Collection, Manassas Va.For a review, see Strathern et al., eds. (1981, The Molecular Biology ofthe Yeast Saccharomyces, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.)

[1042] Methods of introducing exogenous DNA into yeast hosts are wellknown in the art.

[1043] 2. Pichia

[1044] The use of Pichia methanolica as a host cell for the productionof recombinant peptides is disclosed in PCT Applications WO 97/17450, WO97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use intransforming P. methanolica are commonly prepared as double-stranded,circular plasmids, which are preferably linearized prior totransformation. For peptide production in P. methanolica, it ispreferred that the promoter and terminator in the plasmid be that of aP. methanolica gene, such as a P. methanolica alcohol utilization gene(AUG1 or AUG2). Other useful promoters include those of thedihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), andcatalase (CAT) genes, as well as those disclosed in U.S. Pat. No.5,252,726. To facilitate integration of the DNA into the hostchromosome, it is preferred to have the entire expression segment of theplasmid flanked at both ends by host DNA sequences. A preferredselectable marker for use in Pichia methanolica is a P. methanolica ADE2gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC;EC 4.1.1.21), which allows ade2 host cells to grow in the absence ofadenine. For large-scale, industrial processes where it is desirable tominimize the use of methanol, host cells in which both methanolutilization genes (AUG1 and AUG2) are deleted are preferred. Forproduction of secreted peptides, host cells deficient in vacuolarprotease genes (PEP4 and PRB1) are preferred. Electroporation is used tofacilitate the introduction of a plasmid containing DNA encoding apeptide of interest into P. methanolica cells. It is preferred totransform P. methanolica cells by electroporation using an exponentiallydecaying, pulsed electric field having a field strength of from 2.5 to4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from1 to 40 milliseconds, most preferably about 20 milliseconds. For areview of the use of Pichia pastoris for large-scale production ofantibody fragments, see Fischer et al., (1999, Biotechnol Appl Biochem.30 (Pt 2):117-120).

[1045] 3. Aspergillus

[1046] Methods to express peptides in Aspergillus spp. are well known inthe art, including but not limited to those described in Carrez et al.,1990, Gene 94:147-154; Contreras, 1991, Bio/Technology 9:378-381; Yeltonet al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474; Tilburn et al.,1983, Gene 26:205-221; Kelly and. Hynes, 1985, EMBO J. 4:475-479;Ballance et al., 1983, Biochem. Biophys. Res. Comm. 112:284-289; Buxtonet al., 1985, Gene 37:207-214, and U.S. Pat. No. 4,935,349, incorporatedby reference herein in its entirety. Examples of promoters useful inAspergillus are found in U.S. Pat. No. 5,252,726. Strains of Aspergillususeful for peptide expression are found in U.S. Pat. No. 4,935,349.Commercial production of exogenous peptides is available fromNovoenzymes for Aspergillus niger and Aspergillus oryzae.

[1047] 4. Trichoderma

[1048] Trichoderma has certain advantages over other species ofrecombinant host cells for expression of desired peptides. This organismis easy to grow in large quantities and it has the ability toglycosylate and efficiently secrete high yields of recombinant mammalianpeptides into the medium, making isolation of the peptide relativelyeasy. In addition, the glycosylation pattern on expressed peptides ismore similar to that on human peptides than peptides expressed in manyother systems. However, there are still differences in the glycanstructures on expressed peptides from these cells. For example, terminalsialic acid residues are important to the therapeutic function of apeptide in a mammalian system, since the presence of these moieties atthe end of the glycan structure impedes peptide clearance from themammalian bloodstream. The mechanism behind the increased biologichalf-life of sialylated molecules is believed to lie in their decreasedrecognition by lectins (Drickamer, 1988, J. Biol. Chem. 263:9557-9560).However, in general fungal cells do not add terminal sialic acidresidues to glycans on peptides, and peptides synthesized in fungalcells are therefore asialic. According to the present invention, thisdeficiency can be remedied using the in vitro glycan remodeling methodsof the invention described in detail elsewhere herein.

[1049] Trichoderna species useful as hosts for the production ofpeptides to be remodeled include T. reesei, such as QM6a, ALK02442 orCBS383.78 (Centraalbureau voor Schimmelcultures, Oosterstraat 1, PO Box273, 3740 AG Baarn, The Netherlands, or, ATCC13631 (American TypeCulture Collection, Manassas Va., 10852, USA, type); T. viride (such asCBS 189.79 (det. W. Gams); T. longibrachiatum, such as CBS816.68 (type);T. pseudokoningii (such as MUCL19358; Mycotheque de l'UniversiteCatholique de Louvain); T. saturnisporum CBS330.70 (type); T. harzianumCBS316.31 (det. W. Gams); T. virgatum (T. pseudokoningii) ATCC24961.Most preferably, the host is T. reesei and more preferably, it is T.reesei strains QM9414 (ATCC 26921), RUT-C-30 (ATCC 56765), and highlyproductive mutants such as VTT-D-79125, which is derived from QM9414(Nevalainen, Technical Research Centre of Finland Publications 26,(1985), Espoo, Finland).

[1050] The transformation of Trichoderma with DNA is performed using anytechnique known in the art, including that taught in European patent No.EP0244234, Harkki (1989, Bio/Technology 7:596-601) and Uusitalo (1991,J. Biotech. 17:35-50). Culture of Trichoderma is supported by previousextensive experience in industrial scale fermentation techniques; forexample, see Finkelstein, 1992, Biotechnology of Filamentous Fungi:Technology and Products, Butterworth-Heinemann, publishers, Stoneham,Mass.

[1051] 5. Kluyveromyces

[1052] Yeast belonging to the genus Kluyveromyces have been used as hostorganisms for the production of recombinant peptides. Peptides producedby this genus of yeast are, in particular, chymosin (European Patent 96430), thaumatin (European Patent 96 910), albumin, interleukin-1β, TPA,TIMP (European Patent 361 991) and albumin derivatives having atherapeutic function (European Patent 413 622). Species of particularinterest in the genus Kluyveromyces include K. lactis.

[1053] Methods of expressing recombinant peptides in Kluyvermyces spp.are well known in the art. Vectors for the expression and secretion ofhuman recombinant peptides in Kluyvermyces are known in the art (Yeh, J.Cell. Biochem. Suppl. 14C:68, Abst. H402; Fleer, 1990, Yeast 6 (SpecialIssue):S449) as are procedures for transformation and expression ofrecombinant peptides (Ito et al., 1983, J. Bacteriol. 153:163-168; vanden Berg, 1990, Bio/Technology 8:135-139; U.S. Pat. No. 5,633,146,WO8304050A1, EP0096910, EP0241435, EP0301670, EP0361991, all of whichare incorporated by reference herein in their entirety). For a review ofgenetic manipulation of Kluyveromyces lactis linear DNA plasmids by genetargeting and plasmid shuffles, see Schaffrath et al. (1999, FEMSMicrobiol Lett. 178(2):201-210).

[1054] 6. Chrysoporium

[1055] The fungal genus Chrysoporium has recently been used toexpression of foreign recombinant peptides. A description of theproceedures by which one of skill in the art can use Chrysoporium can beused to express foreign peptides is found in WO 00/20555 (incorporatedby reference herein in its entirety). Species particularly suitable forexpression system include, but are not limited to, C. botryoides, C.carmichaelii, C. crassitunicatum, C. europae, C. evolceannui, F.fastidium, C. filiforme, C. gerogiae, C. globiferum, C. globiferum var.articulatum, C. globiferum var. niveum, C. hirundo, C. hispanicum, C.holmii, C. indicum, C. inops, C. keratinophilum, C. kreiselii, C.kuzurovianum, C. lignorum, C. lobatum, C. lucknowense, C. lucknowenseGarg 27K, C. medium, C. medium var. spissescens, C. mephiticum, C.merdarium, C. merdarium var. roseum, C. minor, C. pannicola, C. parvum,C. parvum var. crescens, C. pilosum, C. peodomerderium, C. pyriformis,C. queenslandicum, C. sigleri, C. sulfureum, C. synchronum, C. tropicum,C. undulatum, C. vallenarense, C. vespertilium, and C. zonatum.

[1056] 7. Others

[1057] Methods for transforming Schwanniomyces are disclosed in EuropeanPatent 394 538. Methods for transforming Acremonium chrysogenum aredisclosed by U.S. Pat. No. 5,162,228. Methods for transformingNeurospora are disclosed by U.S. Pat. No. 4,486,533. Also know is anexpression system specifically for Schizosaccharomyces pombe (EuropeanPatent 385 391). General methods for expressing peptides in fissionyeast, Schizosaccharomyces pombe can be found in Giga-Hama and Kumagai(1997, Foreign gene expression in fission yeast: Schizosaccharomycespombe, Springer, Berlin).

[1058] C. Mammalian Systems

[1059] As discussed above, mammalian cells typically produce aheterogeneous mixture of N-glycan structures which vary with respect tothe number and arrangement of additional sugars attached to thetrimannosyl core. Typically, mammalian cells produce peptides having acomplex glycan structure, such as that shown in FIG. 3, right side.Using the methods of the present invention, a peptide produced in amammalian cell may be remodeled in vitro to generate a peptide havingdesired glycosylation by first identifying the primary glycan structureand then determining which sugars must be removed in order to remodelthe glycan structure. As discussed herein, the sugars to be removed willdetermine which cleavage enzymes will be used and thus, the precisesteps of the remodeling process will vary depending on the primaryglycan structure used as the initial substrate. A sample scheme forremodeling a glycan structure commonly produced in mammalian cells isshown in FIG. 2. The N-glycan biosynthetic pathway in mammalian cellshas been well characterized (reviewed in Moremen, 1994, Glycobiology4:113-125). Many of the enzymes necessary for glycan synthesis have beenidentified, and mutant cell lines defective in this enzymatic pathwayhave been isolated including the Chinese hamster ovary (CHO) cell linesLec23 (defective in alpha-glucosidase I) and Lec18 (novel GlcNAc-TVIII).The glycosylation pattern of peptides produced by these mutant cells isaltered relative to normal CHO cells. As discussed herein, theglycosylation defects in these and other mutant cells can be exploitedfor the purposes of producing a peptide that lacks a complex glycanstructure. For example, peptides produced by Lec23 cells lack sialicacid residues, and thus require less enzymatic manipulation in order toreduce the glycan structure to an elemental trimannosyl core or toMan3GlcNAc4. Thus, peptides produced in these cells can serve aspreferred substrates for glycan remodeling. One of ordinary skill in theart could isolate or identify other glycosylation-defective cell linesbased on known methods, for example the method described in Stanley etal., 1990, Somatic Cell Mol. Genet., 16: 211-223. Use ofglycosylation-defective cell lines, those identified and as yetunidentified, is included in the invention for the purpose of generatingpreferred peptide substrates for the remodeling processes describedherein.

[1060] Expression vectors useful for expressing exogenous peptides inmammalian cells are numerous, and are well known to those in the art.Many mammalian expression vectors are now commercially available fromcompanies, including Novagen, Inc (Madison, Wis.), Gene Therapy Systems(San Diego, Calif.), Promega (Madison, Wis.), ClonTech Inc. (Palo Alto,Calif.), and Stratagene (La Jolla, Calif.), among others.

[1061] There are several mammalian cell lines that are particularlyadept at expressing exogenous peptides. Typically mammalian cell linesoriginate from tumor cells extracted from mammals that have becomeimmortalized, that is to say, they can replicate in culture essentiallyindefinitely. These cell lines include, but are not limited to, CHO(Chinese hamster ovary, e.g. CHO-K1; ATCC No. CCL 61) and variantsthereof, NSO (mouse myeloma), BNK, BHK 570 (ATCC No. CRL 10314), BHK(ATCC No. CRL 1632), Per.C6™ (immortalized human cells, Crucell N.V.,Leiden, The Netherlands), COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL1651), HEK 293, mouse L cells, T lymphoid cell lines, BW5147 cells andMDCK (Madin-Darby canine kidney), HeLa (human), A549 (human lungcarcinoma), 293 (ATCC No. CRL 1573; Graham et al., 1977, Gen. Virol.36:59-72), BGMK (Buffalo Green Monkey kidney), Hep-2 (human epidermoidlarynx carcinoma), LLC-MK2 (African Green Monkey Kidney), McCoy,NC1-H292 (human pulmonary mucoepidermoid carcinoma tube), RD(rhabdomyosarcoma), Vero (African Green Monkey kidney), HEL (humanembryonic lung), Human Fetal Lung-Chang, MRC5 (human embryonic lung),MRHF (human foreskin), and WI-38 (human embryonic lung). In some cases,the cells in which the therapeutic peptide is expressed may be cellsderived from the patient to be treated, or they may be derived fromanother related or unrelated mammal. For example, fibroblast cells maybe isolated from the mammal's skin tissue, and cultured and transformedin vitro. This technology is commercially available from TranskaryoticTherapies, Inc. (Cambridge, Mass.). Almost all currently used cell linesare available from the American Type Culture Collection (ATCC, Manassas,Va.) and BioWhittaker (Walkersville, Md.).

[1062] Mammalian cells may be transformed with DNA using any one ofseveral techniques that are well known to those in the art. Suchtechniques include, but are not limited to, calcium phosphatetransformation (Chen and Okayama, 1988; Graham and van der Eb, 1973;Corsaro and Pearson, 1981, Somatic Cell Genetics 7:603),Diethylaminoethyl (DEAE)-dextran transfection (Fujita et al., 1986;Lopata et al., 1984; Selden et al., 1986,), electroporation (Neumann etal., 1982,; Potter, 1988,; Potter et al., 1984,; Wong and Neuman, 1982),cationic lipid reagent transfection (Elroy-Stein and Moss, 1990; Feigneret al., 1987; Rose et al., 1991; Whitt et al., 1990; Hawley-Nelson etal., 1993, Focus 15:73; Ciccarone et al., 1993, Focus 15:80), retroviral(Cepko et al., 1984; Miller and Baltimore, 1986; Pear et al., 1993;Austin and Cepko, 1990; Bodine et al., 1991; Fekete and Cepko, 1993;Lemischka et al., 1986; Turner et al., 1990; Williams et al., 1984;Miller and Rosman, 1989, BioTechniques 7:980-90; Wang and Finer, 1996,Nature Med. 2:714-6), polybrene (Chaney et al, 1986; Kawai andNishizawa, 1984), microinjection (Capecchi, 1980), and protoplast fusion(Rassoulzadegan et al., 1982; Sandri-Goldin et al., 1981; Schaffer,1980), among others. In general, see Sambrook et al. (2001, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York)and Ausubel et al. (2002, Current Protocols in Molecular Biology, JohnWiley & Sons, New York) for transformation techniques.

[1063] Recently the baculovirus system, popular for transformation ofinsect cells, has been adapted for stable transformation of mammaliancells (see, for review, Koat and Condreay, 2002, Trends Biotechnol.20:173-180, and references cited therein). The production of recombinantpeptides in cultured mammalian cells is disclosed, for example, in U.S.Pat. Nos. 4,713,339, 4,784,950; 4,579,821; and 4,656,134. Severalcompanies offer the services of transformation and culture of mammaliancells, including Cell Trends, Inc. (Middletown, Md.). Techniques forculturing mammalian cells are well known in the art, and further foundin Hauser et al. (1997, Mammalian Cell Biotechnology, Walter de Gruyer,Inc., Hawthorne, N.Y.), and Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor and references cited therein.

[1064] D. Insect

[1065] Insect cells and in particular, cultured insect cells, expresspeptides having N-linked glycan structures that are rarely sialylatedand usually comprise mannose residues which may or may not haveadditional fucose residues attached thereto. Examples of the types ofglycan structures present on peptides produced in cultured insect cellsare shown in FIG. 6, and mannose glycans thereof. In this situation,there may or may not be a core fucose present, which if present, may belinked to the glycan via several different linkages.

[1066] Baculovirus-mediated expression in insect cells has becomeparticularly well-established for the production of recombinant peptides(Altmann et al., 1999, Glycoconjugate J. 16:109-123). With regard topeptide folding and post-translational processing, insect cells aresecond only to mammalian cell lines. However, as noted above,N-glycosylation of peptides in insect cells differs in many respectsfrom N-glycosylation in mammalian cells particularly in that insectcells frequently generate truncated glycan structures comprisingoligosaccharides containing just three or sometimes only two mannoseresidues. These structures may be additionally substituted with fucoseresidues.

[1067] According to the present invention, a peptide produced in aninsect cell may be remodeled in vitro to generate a peptide with desiredglycosylation by first optionally removing any substituted fucoseresidues using an appropriate fucosidase enzyme. In instances where thepeptide comprises an elemental trimannosyl core structure following theremoval of fucose residues, then all that is required is the in vitroaddition of the appropriate sugars to the trimannosyl core structure togenerate a peptide having desired glycosylation. In instances when thepeptide might contain only two mannose residues in the glycan structurefollowing removal of any fucose residues, a third mannose residue may beadded using a mannosyltransferase enzyme and a suitable donor moleculesuch as GDP-mannose, and thereafter the appropriate residues are addedto generate a peptide having desired glycosylation. Optionally,monoantennary glycans can also be generated from these species.

[1068] Protocols for the use of baculovirus to transform insect cellsare well known to those in the art. Several books have been publishedwhich provide the procedures to use the baculovirus system to expresspeptides in insect cells. These books include, but are not limited to,Richardson (Baculovirus Expression Protocols, 1998, Methods in MolecularBiology, Vol 39, Humana Pr), O'Reilly et al. (1994, BaculovirusExpression Vectors: A Laboratory Manual, Oxford Univ Press), and Kingand Possee (1992, The Baculovirus Expression System: A Laboratory Guide,Chapman & Hall). In addition, there are also publications such asLucklow (1993, Curr. Opin. Biotechnol. 4:564-572) and Miller (1993,Curr. Opin. Genet. Dev. 3:97-101).

[1069] Many patents have also been issued that related to systems forbaculoviral expression of foreign proteins. These patents include, butare not limited to, U.S. Pat. No. 6,210,966 (Culture medium for insectcells lacking glutamine and containing ammonium salt), U.S. Pat. No.6,090,584 (Use of BVACs (BaculoVirus Artificial Chromosomes) to producerecombinant peptides), U.S. Pat. No. 5,871,986 (Use of a baculovirus toexpress a recombinant nucleic acid in a mammalian cell), U.S. Pat. No.5,759,809 (Methods of expressing peptides in insect cells and methods ofkilling insects), U.S. Pat. No. 5,753,220 (Cysteine protease genedefective baculovirus, process for its production, and process for theproduction of economic peptide by using the same), U.S. Pat. No.5,750,383 (Baculovirus cloning system), U.S. Pat. No. 5,731,182(Non-mammalian DNA virus to express a recombinant nucleic acid in amammalian cell), U.S. Pat. No. 5,728,580 (Methods and culture media forinducing single cell suspension in insect cell lines), U.S. Pat. No.5,583,023 (Modified baculovirus, its preparation process and itsapplication as a gene expression vector), U.S. Pat. No. 5,571,709(Modified baculovirus and baculovirus expression vectors), U.S. Pat. No.5,521,299 (Oligonucleotides for detection of baculovirus infection),U.S. Pat. No. 5,516,657 (Baculovirus vectors for expression of secretoryand membrane-bound peptides), U.S. Pat. No. 5,475,090 (Gene encoding apeptide which enhances virus infection of host insects), U.S. Pat. No.5,472,858 (Production of recombinant peptides in insect larvae), U.S.Pat. No. 5,348,886 (Method of producing recombinant eukaryotic virusesin bacteria), U.S. Pat. No. 5,322,774 (Prokaryotic leader sequence inrecombinant baculovirus expression system), U.S. Pat. No. 5,278,050(Method to improve the efficiency of processing and secretion ofrecombinant genes in insect systems), U.S. Pat. No. 5,244,805(Baculovirus expression vectors), U.S. Pat. No. 5,229,293 (Recombinantbaculovirus), U.S. Pat. No. 5,194,376 (Baculovirus expression systemcapable of producing recombinant peptides at high levels), U.S. Pat. No.5,179,007 (Method and vector for the purification of recombinantpeptides), U.S. Pat. No. 5,169,784 (Baculovirus dual promoter expressionvector), U.S. Pat. No. 5,162,222 (Use of baculovirus early promoters forexpression of recombinant nucleic acids in stably transformed insectcells or recombinant baculoviruses), U.S. Pat. No. 5,155,037 (Insectsignal sequences useful to improve the efficiency of processing andsecretion of recombinant nucleic acids in insect systems), U.S. Pat. No.5,147,788 (Baculovirus vectors and methods of use), U.S. Pat. No.5,110,729 (Method of producing peptides using baculovirus vectors incultured cells), U.S. Pat. No. 5,077,214 (Use of baculovirus earlypromoters for expression of recombinant genes in stably transformedinsect cells), U.S. Pat. No. 5,023,328 (Lepidopteran AKH signalsequence), and U.S. Pat. Nos. 4,879,236 and 4,745,051 (Method forproducing a recombinant baculovirus expression vector). All of theaforementioned patents are incorporated in their entirety by referenceherein.

[1070] Insect cell lines of several different species origin arecurrently being used for peptide expression, and these lines are wellknown to those in the art. Insect cell lines of interest include, butare not limited to, dipteran and lepidopteran insect cells in general,Sf9 and variants thereof (fall armyworm Spodoptera frugiperda),Estigmene acrea, Trichoplusia ni, Bombyx mori, Malacosoma disstri.drosophila lines Kc1 and SL2 among others, and mosquito.

[1071] E. Plants

[1072] Plant cells as peptide producers present a different set ofissues. While N-linked glycans produced in plants comprise a trimannosylcore structure, this pentasaccharide backbone may comprise severaldifferent additional sugars as shown in FIG. 5. For example, in oneinstance, the trimannosyl core structure is substituted by a β1,2 linkedxylose residue and an α1,3 linked fucose residue. In addition, plantcells may also produce a Man5GlcNAc2 structure. Peptides produced inplant cells are often highly antigenic as a result of the presence ofthe core α1,3 fucose and xylose on the glycan structure, and are rapidlycleared from the blood stream when introduced into a mammal due to theabsence of terminal sialic acid residues. Therefore, unless thesepeptides are remodeled using the methods provided herein, they aregenerally considered to be unsuitable as therapeutic agents in mammals.While some monoclonal antibodies expressed in plant cells were found tobe non-immunogenic in mouse, it is likely that the glycan chains werenot immunogenic because they were buried in the Fc region in theseantibodies (Chargelegue et al., 2000, Transgenic Res. 9(3):187-194).

[1073] Following the directions provided herein, it is now possible togenerate a peptide produced in a plant cell wherein an increased numberof the glycan structures present thereon comprise an elementaltrimannosyl core structure, or a Man3GlcNAc4 structure. This isaccomplished by cleaving off any additional sugars in vitro using acombination of appropriate glycosidases, including fucosidases, untilthe elemental trimannosyl core structure or the Man3GlcNAc4 structure isarrived at. These cleavage reactions should also include removal of anyfucose or xylose residues from the structures in order to diminish theantigenicity of the final peptide when introduced into a mammal. Plantcells having mutations that inhibit the addition of fucose and xyloseresidues to the trimannosyl core structure are known in the art (vonSchaewen et al., 1993, Plant Physiology 102:1109-1118). The use of thesecells to produce peptides having glycans which lack fucose and xylose iscontemplated by the invention. Upon production of the elementaltrimannosyl core or Man3GlcNAc4 structure, additional sugars may then beadded thereto to arrive at a peptide having desired glycosylation thatis therefore suitable for therapeutic use in a mammal.

[1074] Transgenic plants are considered by many to be the expressionsystem of choice for pharmaceutical peptides. Potentially, plants canprovide a cheaper source of recombinant peptides. It has been estimatedthat the production costs of recombinant peptides in plants could bebetween 10 to 50 times lower that that of producing the same peptide inE. coli. While there are slight differences in the codon usage in plantsas compared to animals, these can be compensated for by adjusting therecombinant DNA sequences (see, Kusnadi et al., 1997, Biotechnol.Bioeng. 56:473-484; Khoudi et al., 1999, Biotechnol. Bioeng. 135-143;Hood et al., 1999, Adv. Exp. Med. Biol. 464:127-147). In addition,peptide synthesis, secretion and post-translational modification arevery similar in plants and animals, with only minor differences in plantglycosylation (see, Fischer et al., 2000, J. Biol. Regul. Homest. Agents14: 83-92). Then, products from transgenic plants are also less likelyto be contaminated by animal pathogens, microbial toxins and oncogenicsequences.

[1075] The expression of recombinant peptides in plant cells is wellknown in the art. In addition to transgenic plants, peptides can alsoproduced in transgenic plant cell cultures (Lee et al., 1997, Mol. Cell.7:783-787), and non-transgenic plants inoculated with recombinant plantviruses. Several books have been published that describe protocols forthe genetic transformation of plant cells: Potrykus (1995, Gene transferto plants, Springer, New York), Nickoloff (1995, Plant cellelectroporation and electrofusion protocols, Humana Press, Totowa, N.Y.)and Draper (1988, Plant genetic transformation, Oxford Press, Boston).

[1076] Several methods are currently used to stably transform plantcells with recombinant genetic material. These methods include, but arenot limited to, Agrobacterium transformation (Bechtold and Pelletier,1998; Escudero and Hohn, 1997; Hansen and Chilton, 1999; Touraev et al.,1997), biolistics (microprojectiles) (Finer et al., 1999; Hansen andChilton, 1999; Shilito, 1999), electroporation of protoplasts (Fromm etal., 1985, Ou-Lee et al., 1986; Rhodes et al., 1988; Saunders et al.,1989; Trick et al., 1997), polyethylene glycol treatment (Shilito, 1999;Trick et al., 1997), in planta mircroinjection (Leduc et al., 1996; Zhouet al., 1983), seed imbibition (Trick et al., 1997), laser beam (1996),and silicon carbide whiskers (Thompson et al., 1995; U.S. Patent Appln.No. 20020100077, incorporated by reference herein in its entirety).

[1077] Many kinds of plants are amenable to transformation andexpression of exogenous peptides. Plants of particular interest toexpress the peptides to be used in the remodeling method of theinvention include, but are not limited to, Arabidopsis thalliana,rapeseed (Brassica spp.; Ruiz and Blumwald, 2002, Planta 214:965-969)),soybean (Glycine max), sunflower (Helianthus unnuus), oil palm (Elaeisguineeis), groundnut (peanut, Arachis hypogaea; Deng et al., 2001, Cell.Res. 11: 156-160), coconut (Cocus nucifera), castor (Ricinus communis),safflower (Carthamus tinctorius), mustard (Brassica spp. and Sinapisalba), coriander, (Coriandrum sativum), squash (Cucurbita maxima;Spencer and Snow, 2001, Heredity 86(Pt 6):694-702), linseed/flax (Linumusitatissimum; Lamblin et al., 2001, Physiol Plant 112:223-232), Brazilnut (Bertholletia excelsa), jojoba (Simmondsia chinensis), maize (Zeamays; Hood et al., 1999, Adv. Exp. Med. Biol. 464:127-147; Hood et al.,1997, Mol. Breed. 3:291-306; Petolino et al., 2000, Transgenic Research9:1-9), alfalfa (Khoudi et al., 1999, Biotechnol. Bioeng. 64:135-143),tobacco (Nicotiana tabacum; Wright et al., Transgenic Res. 10:177-181;Frigerio et al., 2000, Plant Physiol. 123:1483-1493; Cramer et al.,1996, Ann. New York Acad. Sci. 792:62-8-71; Cabanes-Macheteau et al.,1999, Glycobiology 9:365-372; Ruggiero et al., 2000, FEBS Lett.469:132-136), canola (Bai et al., 2001, Biotechnol. Prog. 17:168-174;Zhang et al., 2000, J. Anim. Sci. 78:2868-2878)), potato (Tacket et al.,1998, J. Infect. Dis. 182:302-305; Richter et al., 2000, Nat.Biotechnol. 18:1167-1171; Chong et al., 2000, Transgenic Res. 9:71-78),alfalfa (Wigdorovitz et al., 1999, Virology 255:347-353), Pea (Pisumsativum; Perrin et al., 2000, Mol. Breed. 6:345-352), rice (Oryzasativa; Stoger et al., 2000, Plant Mol. Biol. 42:583-590), cotton(Gossypium hirsutum; Kornyeyev et al., 2001, Physiol Plant 113:323-331),barley (Hordeum vulgare; Petersen et al., 2002, Plant Mol Biol49:45-58); wheat (Triticum spp.; Pellegrineschi et al., 2002, Genome45:421-430) and bean (Vicia spp.; Saalbach et al., 1994, Mol Gen Genet242:226-236).

[1078] If expression of the recombinant nucleic acid is desired in awhole plant rather than in cultured cells, plant cells are firsttransformed with DNA encoding the peptide, following which, the plant isregenerated. This involves tissue culture procedures that are typicallyoptimized for each plant species. Protocols to regenerate plants arealready well known in the art for many species. Furthermore, protocolsfor other species can be developed by one of skill in the art usingroutine experimentation. Numerous laboratory manuals are available thatdescribe procedures for plant regeneration, including but not limitedto, Smith (2000, Plant tissue culture: techniques and experiments,Academic Press, San Diego), Bhojwani and Razdan (1996, Plant tissueculture: theory and practice, Elsevier Science Pub., Amsterdam), Islam(1996, Plant tissue culture, Oxford & IBH Pub. Co., New Delhi, India),Dodds and. Roberts (1995, Experiments in plant tissue culture, New York:Cambridge University Press, Cambridge England), Bhojwani (Plant tissueculture: applications and limitations, Elsevier, Amsterdam, 1990),Trigiano and Gray (2000, Plant tissue culture concepts and laboratoryexercises, CRC Press, Boca Raton, Fla.), and Lindsey (1991, Plant tissueculture manual: fundamentals and applications, Kluwer Academic, Boston).

[1079] While purifying recombinant peptides from plants may potentiallybe costly, several systems have been developed to minimize these costs.One method directs the synthesized peptide to the seed endosperm fromwhere it can easily extracted (Wright et al., 2001, Transgenic Res.10:177-181, Guda et al., 2000, Plant Cell Res. 19:257-262; and U.S. Pat.No. 5,767,379, which is incorporated by reference herein in itsentirety). An alternative approach is the co-extraction of therecombinant peptide with conventional plant products such as starch,meal or oil. In oil-seed rape, a fusion peptide of oleosin-hurudin whenexpressed in the plant, attaches to the oil body of the seed, and can beextracted from the plant seed along with the oil (Parmenter, 1995, PlantMol. Biol. 29:1167-1180;. U.S. Pat. Nos. 5,650,554, 5,792,922, 5,948,682and 6,288,304, and US application 2002/0037303, all of which areincorporated in their entirely by reference herein). In a variation onthis approach, the oleosin is fused to a peptide having affinity for theexogenous co-expressed peptide of interest (U.S. Pat. No. 5,856,452,incorporated by reference herein in its entirety).

[1080] Expression of recombinant peptides in plant plastids, such as thechloroplast, generates peptides having no glycan structures attachedthereto, similar to the situation in prokaryotes. However, the yield ofsuch peptides is vastly greater when expressed in these plant cellorganelles, and thus this type of expression system may have advantagesover other systems. For a general review on the technology for plastidexpression of exogenous peptides in higher plants, see Hager and Beck(2000, Appl. Microbiol. Biotechnol. 54:302-310, and references citedtherein). Plastid expression has been particularly successful in tobacco(see, for example, Staub et al., 2000, Nat. Biotechnol. 18:333-338).

[1081] F. Transgenic Animals

[1082] Introduction of a recombinant DNA into the fertilized egg of ananimal (e.g., a mammal) may be accomplished using any number of standardtechniques in transgenic animal technology. See, e.g., Hogan et al.,Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986; and U.S. Pat. No.5,811,634, which is incorporated by reference herein in its entirety.Most commonly, the recombinant DNA is introduced into the embryo by wayof pronuclear microinjection (Gordon et al., 1980, PNAS 77:7380-7384;Gordon and Ruddle, 1981, Science 214:1244-1246; Brinster et al., 1981,Cell 27:223-231; Costantini and Lacy, 1981, Nature 294:92-94).Microinjection has the advantage of being applicable to a wide varietyof species. Preimplantation embryos may also be transformed withretroviruses (Jaenisch and Mintz, 1974, Proc. Natl. Acad. Sci. U.S.A.71:1250-1254; Jaenisch et al., 1976, Hamatol Bluttransfus. 19:341-356;Stuhlmann et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:7151-7155).Retroviral mediated transformation has the advantage of adding singlecopies of the recombinant nucleic acid to the cell, but it produces ahigh degree of mosaicism. Most recently, embryonic stem cell-mediatedtechniques have been used (Gossler et al., 1986, Proc. Natl. Acad. Sci.U.S.A. 83:9065-9069), transfer of entire chromosomal segments (Lavitranoet al., 1989, Cell 57:717-723), and gamete transfection in conjunctionwith in vitro fertilization (Lavitrano et al., 1989, Cell 57:717-723)have also been used. Several books of laboratory procedures have beenpublished disclosing these techniques: Cid-Arregui and García-Carrancá(1998, Microinjection and Transgenesis: Strategies and Protocols,Springer, Berlin), Clarke (2002, Transgenesis Techniques: Principles andProtocols, Humana Press, Totowa, N.J.), and Pinkert (1994, TransgenicAnimal Technology: A Laboratory Handbook, Academic Press, San Diego).

[1083] Once the recombinant DNA is introduced into the egg, the egg isincubated for a short period of time and is then transferred into apseudopregnant animal of the same species from which the egg wasobtained (Hogan et al., supra). In the case of mammals, typically 125eggs are injected per experiment, approximately two-thirds of which willsurvive the procedure. Twenty viable eggs are transferred into apseudopregnant mammal, four to ten of which will develop into liveprogeny. Typically, 10-30% of the progeny (in the case of mice) carrythe recombinant DNA.

[1084] While the entire animal can be used as an expression system forthe peptides of the invention, in a preferred embodiment, the exogenouspeptide accumulates in products of the animal, from which it can beharvested without injury to the animal. In preferred embodiments, theexogenous peptide accumulates in milk, eggs, hair, blood, and urine.

[1085] If the recombinant peptide is to be accumulated in the milk ofthe animal, suitable mammals are ruminants, ungulates, domesticatedmammals, and dairy animals. Particularly preferred animals are goats,sheep, camels, cows, pigs, horses, oxen, and llamas. Methods forgenerating transgenic cows that accumulate a recombinant peptide intheir milk are well known: see, Newton (1999, J. Immunol. Methods231:159-167), Ebert et al. (1991, Biotechnology 9: 835-838), and U.S.Pat. Nos. 6,210,736, 5,849,992, 5,843,705, 5,827,690, 6,222,094, all ofwhich are incorporated herein by reference in their entirety. Thegeneration of transgenic mammals that produce a desired recombinantpeptide is commercially available from GTC Biotherapeutics, Framingham,Mass.

[1086] If the recombinant peptide is to be accumulated in eggs, suitablebirds include, but are not limited to, chickens, geese, and turkeys.Other animals of interest include, but are not limited to, other speciesof avians, fish, reptiles and amphibians. The introduction ofrecombinant DNA to a chicken by retroviral transformation is well knownin the art: Thoraval et al. (1995, Transgenic Research 4:369-376),Bosselman et al., (1989, Science 243: 533-535), Petropoulos et al.(1992, J. Virol. 66: 3391-3397), U.S. Pat. No. 5,162,215, incorporatedby reference herein in its entirety. Successful transformation ofchickens with recombinant DNA also been achieved wherein DNA isintroduced into blastodermal cells and blastodermal cells so transfectedare introduced into the embryo: Brazolot et al. (1991, Mol. Reprod. Dev.30: 304-312), Fraser, et al. (1993, Int. J. Dev. Biol. 37: 381-385), andPetitte et al. (1990, Development 108: 185-189). High throughputtechnology has been developed to assess whether a transgenic chickenexpresses the desired peptide (Harvey et al., 2002, Poult. Sci.81:202-212, U.S. Pat. No. 6,423,488, incorporated by reference herein inits entirety). Using retroviral transformation of chicken with arecombinant DNA, exogenous beta-lactamase was accumulated in the eggwhite of the chicken (Harvey et al., 2002, Nat. Biotechnol.20(4):396-399). The production of chickens producing exogenous peptidesin egg is commercially available from AviGenics, Inc., Athens Ga.

[1087] G. Bacteria

[1088] Recombinantly expressed peptides produced in bacteria are notgenerally glycosylated. However, bacteria systems capable ofglycosylating peptides are becoming evident and therefore it is likelythat glycosylated recombinant peptides may be produced in bacteria inthe future.

[1089] Numerous bacterial expression systems are known in the art.Preferred bacterial species include, but are not limited to, E. coli.and Bacillus species. The expression of recombinant peptides in E. coliis well known in the art. Protocols for E. coli-based expression systemsare found in U.S. Appln No. 20020064835, U.S. Pat. Nos. 6,245,539,5,606,031, 5,420,027, 5,151,511, and RE33,653, among others. Methods totransform bacteria include, but are not limited to, calcium chloride(Cohen et al., 1972, Proc. Natl. Acad. Sci. U.S.A. 69:2110-2114;Hanahan, 1983, J. Mol. Biol. 166:557-580; Mandel and Higa, 1970, J. Mol.Biol. 53:159-162) and electroporation (Shigekawa and Dower, 1988,Biotechniques 6:742-751), and those described in Sambrook et al., 2001(supra). For a review of laboratory protocols on microbialtransformation and expression systems, see Saunders and Saunders (1987,Microbial Genetics Applied to Biotechnology: Principles and Techniquesof Gene Transfer and Manipulation, Croom Helm, London), Pühler (1993,Genetic Engineering of Microorganisms, Weinheim, N.Y.), Lee et al.,(1999, Metabolic Engineering, Marcel Dekker, New York), Adolph (1996,Microbial Genome Methods, CRC Press, Boca Raton), and Birren and Lai(1996, Nonmammalian Genomic Analysis: A Practical Guide, Academic Press,San Diego),

[1090] For a general review on the literature for peptide expression inE. coli see Balbas (2001, Mol. Biotechnol. 19:251-267). Severalcompanies now offer bacterial strains selected for the expression ofmammalian peptides, such as the Rosetta™ strains of E. coli (Novagen,inc., Madison, Wis.; with enhanced expression of eukaryotic codons notnormally used in bacteria cells, and enhanced disulfide bond formation),

[1091] H. Cell Engineering

[1092] It will be apparent from the present disclosure that the moreuniform the starting material produced by a cell, the more efficientwill be the generation in vitro of large quantities of peptides havingdesired glycosylation. Thus, the genetic engineering of host cells toproduce uniformly glycosylated peptides as starting material for the invitro enzymatic reactions disclosed herein, provides a significantadvantage over using a peptide starting material having a heterogeneousset of glycan structures attached thereto. One preferred peptidestarting material for use in the present invention is a peptide havingprimarily glycan molecules which consist solely of an elementaltrimannosyl core structure. Another preferred starting material isMan3GlcNAc4. Following the remodeling process, the preferred peptideswill give rise to the greatest amount of peptides having desiredglycosylation, and thus improved clinical efficacy. However, otherglycan starting material is also suitable for use in the methodsdescribed herein, in that for example, high mannose glycans may beeasily reduced, in vitro, to elemental trimannosyl core structures usinga series of mannosidases. As described elsewhere herein, other glycanstarting material may also be used, provided it is possible to cleaveoff all extraneous sugar moieties so that the elemental trimannosyl corestructure or Man3GlcNAc4 is generated. Thus, the purpose of usinggenetically engineered cells for the production of the peptides of thepresent invention is to generate peptides having as uniform as possiblea glycan structure attached thereto, wherein the glycan structure can beremodeled in vitro to generate a peptide having desired glycosylation.This will result in a dramatic reduction in production costs of thesepeptides. Since the glycopeptides produced using this methodology willpredominantly have the same N-linked glycan structure, thepost-production modification protocol can be standardized and optimizedto produce a greater batch-to-batch consistency of final product. As aresult, the final completed-chain products may be less heterogeneousthan those presently available. The products will have an improvedbiological half-life and bioactivity as compared to the products of theprior art. Alternatively, if desired, the invention can be used tointroduce limited and specific heterogeneity, e.g., by choosing reactionconditions that result in differential addition of sugar moieties.

[1093] Preferably, though not as a rigid requirement, the geneticallyengineered cell is one which produces peptides having glycan structurescomprised primarily of an elemental trimannosyl core structure orMan3GlcNAc4. At a minimum, the proportion of these preferred structuresproduced by the genetically engineered cell must be enough to yield apeptide having desired glycosylation following the remodeling protocol.

[1094] In general, any eukaryotic cell type can be modified to become ahost cell of the present invention. First, the glycosylation pattern ofboth endogenous and recombinant glycopeptides produced by the organismare determined in order to identify suitable additions/deletions ofenzymatic activities that result in the production of elementaltrimannosyl core glycopeptides or Man3GlcNAc4 glycopeptides. This willtypically entail deleting activities that use trimannosyl glycopeptidesas substrates for a glycosyltransferase reaction and inserting enzymaticactivities that degrade more complex N-linked glycans to produce shorterchains. In addition, genetically engineered cells may produce highmannose glycans, which may be cleaved by mannosidase to produce desiredstarting glycan structures. The mannosidase may be active in vivo in thecell (i.e., the cell may be genetically engineered to produce them), orthey may be used in in vitro post production reactions.

[1095] Techniques for genetically modifying host cells to alter theglycosylation profile of expressed peptides are well-known. See, e.g.,Altmann et al. (1999, Glycoconjugate J. 16: 109-123), Ailor et al.(2000, Glycobiology 10(8): 837-847), Jarvis et al., (In vitrogenConference, March, 1999, abstract), Hollister and Jarvis, (2001,Glycobiology 11(1): 1-9), and Palacpac et al., (1999, PNAS USA 96:4697), Jarvis et al., (1998. Curr. Opin. Biotechnol. 9:528-533),Gemgross (U.S. Patent Publication No. 20020137134), all of whichdisclose techniques to “mammalianize” insect or plant cell expressionsystems by transfecting insect or plant cells with glycosyltransferasegenes.

[1096] Techniques also exist to genetically alter the glycosylationprofile of peptides expressed in E. coli. E. coli has been engineeredwith various glycosyltransferases from the bacteria Neisseriameningitidis and Azorhizobium to produce oligosaccharides in vivo(Bettler et al., 1999, Glycoconj. J. 16:205-212). E. coli which has beengenetically engineered to over-express Neisseria meningitidis β1,3 Nacetyl glucosaminyltransferase IgtA gene will efficiently glycosylateexogenous lactose (Priem et al., 2002, Glycobiology 12:235-240).

[1097] Fungal cells have also been genetically modified to produceexogenous glycosyltransferases (Yoshida et al., 1999, Glycobiology,9(1):53-58; Kalsner et al., 1995, Glycoconj. J. 12:360-370; Schwientekand Ernst, 1994, Gene 145(2):299-303; Chiba et al, 1995, Biochem J.308:405-409).

[1098] Thus, in one aspect, the present invention provides a cell thatglycosylates a glycopeptide population such that a proportion ofglycopeptides produced thereby have an elemental trimannosyl core or aMan3GlcNAc4 structure. Preferably, the cell produces a peptide having aglycan structure comprised solely of an elemental trimannosyl core. At aminimum, the proportion of peptides having an elemental trimannosyl coreor a Man3GlcNAc4 structure is enough to yield peptides having desiredglycosylation following the remodeling process. The cell has introducedinto it one or more heterologous nucleic acid expression units, each ofwhich may comprise one or more nucleic acid sequences encoding one ormore peptides of interest. The natural form of the glycopeptide ofinterest may comprise one or more complex N-linked glycans or may simplybe a high mannose glycan.

[1099] The cell may be any type of cell and is preferably a eukaryoticcell. The cell may be a mammalian cell such as human, mouse, rat,rabbit, hamster or other type of mammalian cell. When the cell is amammalian cell, the mammalian cell may be derived from or containedwithin a non-human transgenic mammal where the cell in the mammalencodes the desired glycopeptide and a variety of glycosylating andglycosidase enzymes as necessary for the production of desiredglycopeptide molecules. In addition, the cell may be a fungal cell,preferably, a yeast cell, or the cell may be an insect or a plant cell.Similarly, when the cell is a plant cell, the plant cell may be derivedfrom or contained within a transgenic plant, wherein the plant encodesthe desired glycopeptide and a variety of glycosylating and glycosidaseenzymes as are necessary for the production of desired glycopeptidemolecules.

[1100] In some embodiments the host cell may be a eukaryotic cellexpressing one or more heterologous glycosyltransferase enzymes and/orone or more heterologous glycosidase enzymes, wherein expression of arecombinant glycopeptide in the host cell results in the production of arecombinant glycopeptide having an elemental trimannosyl core as theprimary glycan structure attached thereto.

[1101] In some embodiments the heterologous glycosyltransferase enzymeuseful in the cell may be selected from a group consisting of any knownglycosyltransferase enzyme included for example, in the list ofGlycosyltransferase Families available in Taniguchi et al. (2002,Handbook of Glycosyltransferases and Related Genes, Springer, New York).

[1102] In other embodiments, the heterologous glycosylase enzyme may beselected from a group consisting of mannosidase 1, mannosidase 2,mannosidase 3, and other mannosidases, including, but not limited to,microbial mannosidases. Additional disclosure regarding enzymes usefulin the present invention is provided elsewhere herein.

[1103] In yet other embodiments, the host cell may be a eukaryotic cellwherein one or more endogenous glycosyltransferase enzymes and/or one ormore endogenous glycosidase enzymes have been inactivated such thatexpression of a recombinant glycopeptide in the host cell results in theproduction of a recombinant glycopeptide having an elemental trimannosylcore as the primary glycan structure attached thereto.

[1104] In additional embodiments, the host cell may express heterologousglycosyltransferase enzymes and/or glycosidase enzymes while at the sametime one or more endogenous glycosyltransferase enzymes and/orglycosidase enzymes are inactivated. Endogenous glycosyltransferaseenzymes and/or glycosidase enzymes may be inactivated using anytechnique known to those skilled in the art including, but not limitedto, antisense techniques and techniques involving insertion of nucleicacids into the genome of the host cell. In some embodiments, theendogenous enzymes may be selected from a group consisting of GnT-I, aselection of mannosidases, xylosyltransferase, core α1,3fucosyltransferase, serine/threonine O-mannosyltransferases, and thelike.

[1105] Alternatively, an expression system that naturally glycosylatespeptides such that the N-linked glycans are predominantly thetrimannosyl core type, or the Man3GlcNAc4 type, can be exploited. Anexample of a cell type that produces the trimannosyl core is Sf9 cells.Other such expression systems can be identified by analyzingglycopeptides that are naturally or recombinantly expressed in cells andselecting those which exhibit the desired glycosylation characteristics.The invention should be construed to include any and all such cells forthe production of the peptides of the present invention.

[1106] V. Purification of Glycan Remodeled and/or GlycoconjugatedPeptides

[1107] If the modified glycoprotein is produced intracellularly orsecreted, as a first step, the particulate debris, either host cells,lysed fragments, is removed, for example, by centrifugation orultrafiltration; optionally, the protein may be concentrated with acommercially available protein concentration filter, followed byseparating the peptide variant from other impurities by one or moresteps selected from immunoaffinity chromatography, ion-exchange columnfractionation (e.g., on diethylaminoethyl (DEAE) or matrices containingcarboxymethyl or sulfopropyl groups), chromatography on Blue-Sepharose,CM Blue-Sepharose, MONO-Q, MONO—S, lentil lectin-Sepharose,WGA-Sepharose, Con A-Sepharose, Ether Toyopearl, Butyl Toyopearl, PhenylToyopearl, or protein A Sepharose, SDS-PAGE chromatography, silicachromatography, chromatofocusing, reverse phase HPLC (RP-HPLC), gelfiltration using, e.g., Sephadex molecular sieve or size-exclusionchromatography, chromatography on columns that selectively bind thepeptide, and ethanol, pH or ammonium sulfate precipitation, membranefiltration and various techniques.

[1108] Modified peptides produced in culture are usually isolated byinitial extraction from cells, enzymes, etc., followed by one or moreconcentration, salting-out, aqueous ion-exchange, or size-exclusionchromatography steps. Additionally, the modified glycoprotein may bepurified by affinity chromatography. Then, HPLC may be employed forfinal purification steps.

[1109] A protease inhibitor, e.g., phenylmethylsulfonylfluoride (PMSF)may be included in any of the foregoing steps to inhibit proteolysis andantibiotics may be included to prevent the growth of adventitiouscontaminants.

[1110] Within another embodiment, supernatants from systems whichproduce the modified peptide of the invention are first concentratedusing a commercially available protein concentration filter, forexample, an Amicon or Millipore Pellicon ultrafiltration unit. Followingthe concentration step, the concentrate may be applied to a suitablepurification matrix. For example, a suitable affinity matrix maycomprise a ligand for the peptide, a lectin or antibody molecule boundto a suitable support. Alternatively, an anion-exchange resin may beemployed, for example, a matrix or substrate having pendant DEAE groups.Suitable matrices include acrylamide, agarose, dextran, cellulose, orother types commonly employed in protein purification. Alternatively, acation-exchange step may be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Sulfopropyl groups are particularly preferred.

[1111] Then, one or more RP-HPLC steps employing hydrophobic RP-HPLCmedia, e.g., silica gel having pendant methyl or other aliphatic groups,may be employed to further purify a peptide variant composition. Some orall of the foregoing purification steps, in various combinations, canalso be employed to provide a homogeneous modified glycoprotein.

[1112] The modified peptide of the invention resulting from alarge-scale fermentation may be purified by methods analogous to thosedisclosed by Urdal et al., J. Chromatog. 296: 171 (1984). This referencedescribes two sequential, RP-HPLC steps for purification of recombinanthuman IL-2 on a preparative HPLC column. Alternatively, techniques suchas affinity chromatography may be utilized to purify the modifiedglycoprotein.

[1113] VI. Preferred Peptides and Nucleic Acids Encoding PreferredPeptides

[1114] The present invention includes isolated nucleic acids encodingvarious peptides and proteins, and similar molecules or fragmentsthereof. The invention should not be construed to be limited in any waysolely to the use of these peptides in the methods of the invention, butrather should be construed to include any and all peptides presentlyavailable or which become available to those in the art. In addition,the invention should not be construed to include only one particularnucleic acid or amino acid sequence for the peptides listed herein, butrather should be construed to include any and all variants, homologs,mutants, etc. of each of the peptides. It should be noted that when aparticular peptide is identified as having a mutation or otheralteration in the sequence for that peptide, the numbering of the aminoacids which identify the alteration or mutation is set so that the firstamino acid in the mature peptide sequence is amino acid no. 1, unlessotherwise stated herein.

[1115] Preferred peptides include, but are not limited to humangranulocyte colony stimulating factor (G-CSF), human interferon alpha(IFN-alpha), human interferon beta (IFN-beta), human Factor VII (FactorVII), human Factor IX (Factor IX), human follicle stimulating hormone(FSH), human erythropoietin (EPO), human granulocyte/macrophage colonystimulating factor (GM-CSF), human interferon gamma (IFN-gamma), humanalpha-1-protease inhibitor (also known as alpha-1-antitrypsin oralpha-1-trypsin inhibitor; A-1-PI), glucocerebrosidase, humantissue-type activator (TPA), human interleukin-2 (IL-2), human FactorVIII (Factor VIII), a 75 kDa tumor necrosis factor receptor fused to ahuman IgG immunoglobulin Fc portion, commercially known as ENBREL™ orETANERCEPT™ (chimeric TNFR), human urokinase (urokinase), a Fab fragmentof the human/mouse chimeric monoclonal antibody that specifically bindsglycoprotein IIb/IIIa and the vitronectin alpha_(V) beta₃ receptor,known commercially as REOPRO™ or ABCIXIMAB (chimeric anti-glycoproteinIIb/IIIa), a mouse/human chimeric monoclonal antibody that specificallybinds human HER2, known commercially as HERCEPTIN™ (chimeric anti-HER2),a human/mouse chimeric antibody that specifically binds the A antigenicsite or the F protein of respiratory syncytial virus commercially knownas SYNAGIS™ or PALIVIZUMAB (chimeric anti-RSV), a chimeric human/mousemonoclonal antibody that specifically binds CD20 on human B-cells, knowncommercially as RITUXAN™ or RITUXAMAB (chimeric anti-CD20), humanrecombinant DNase (DNase), a chimeric human/mouse monoclonal antibodythat specifically binds human tumor necrosis factor, known commerciallyas REMICADE™ or INFLIXIMAB (chimeric anti-TNF), human insulin, thesurface antigen of a hepatitis B virus (adw subtype; HBsAg), and humangrowth hormone (HGH), alpha-galactosidase A (Fabryzyme™), α-Iduronidase(Aldurazyme™), antithrombin (antithrombin III, AT-III), human chorionicgonadotropin (hCG), interferon omega, and the like.

[1116] The isolated nucleic acid of the invention should be construed toinclude an RNA or a DNA sequence encoding any of the above-identifiedpeptides of the invention, and any modified forms thereof, includingchemical modifications of the DNA or RNA which render the nucleotidesequence more stable when it is cell free or when it is associated witha cell. As a non-limiting example, oligonucleotides which contain atleast one phosphorothioate modification are known to confer upon theoligonucleotide enhanced resistance to nucleases. Specific examples ofmodified oligonucleotides include those which contain phosphorothioate,phosphotriester, methyl phosphonate, short chain alkyl or cycloalkylintersugar linkages, or short chain heteroatomic or heterocyclicintersugar (“backbone”) linkages. In addition, oligonucleotides havingmorpholino backbone structures (U.S. Pat. No. 5,034,506) or polyamidebackbone structures (Nielsen et al., 1991, Science 254: 1497) may alsobe used.

[1117] Chemical modifications of nucleotides may also be used to enhancethe efficiency with which a nucleotide sequence is taken up by a cell orthe efficiency with which it is expressed in a cell. Any and allcombinations of modifications of the nucleotide sequences arecontemplated in the present invention.

[1118] The present invention should not be construed as being limitedsolely to the nucleic and amino acid sequences disclosed herein. Asdescribed in more detail elsewhere herein, once armed with the presentinvention, it is readily apparent to one skilled in the art that othernucleic acids encoding the peptides of the present invention can beobtained by following the procedures described herein (e.g.,site-directed mutagenesis, frame shift mutations, and the like), andprocedures that are well-known in the art.

[1119] Also included are isolated nucleic acids encoding fragments ofpeptides, wherein the peptide fragments retain the desired biologicalactivity of the peptide. In addition, although exemplary nucleic acidsencoding preferred peptides are disclosed herein in relation to specificSEQ ID NOS, the invention should in no way be construed to be limited toany specific nucleic acid disclosed herein. Rather, the invention shouldbe construed to include any and all nucleic acid molecules having asufficient percent identity with the sequences disclosed herein suchthat these nucleic acids also encode a peptide having the desiredbiological activity disclosed herein. Also contemplated are isolatednucleic acids that are shorter than full length nucleic acids, whereinthe biological activity of the peptide encoded thereby is retained.Methods to determine the percent identity between one nucleic acid andanother are disclosed elsewhere herein as are assays for thedetermination of the biological activity of any specific preferredpeptide.

[1120] Also as disclosed elsewhere herein, any other number ofprocedures may be used for the generation of derivative, mutant, orvariant forms of the peptides of the present invention using recombinantDNA methodology well known in the art such as, for example, thatdescribed in Sambrook et al. (1989, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, New York) and Ausubel etal. (1997, Current Protocols in Molecular Biology, Green & Wiley, NewYork). Procedures for the introduction of amino acid changes in apeptide or polypeptide by altering the DNA sequence encoding the peptideare well known in the art and are also described in Sambrook et al.(1989, supra); Ausubel et al. (1997, supra).

[1121] The invention includes a nucleic acid encoding a G-CSF,IFN-alpha, IFN-beta, Factor VII, Factor IX, FSH, EPO, GM-CSF, IFN-gamma,A-1-PI, glucocerebrosidase, TPA, IL-2, Factor VIII, chimeric TNFR,urokinase, chimeric anti-glycoprotein IIb/IIIa, chimeric anti-HER2,chimeric anti-RSV, chimeric anti-CD20, DNase, chimeric anti-TNF, humaninsulin, HBsAg, and HGH, wherein a nucleic acid encoding a tag peptideis covalently linked thereto. That is, the invention encompasses achimeric nucleic acid wherein the nucleic acid sequence encoding a tagpeptide is covalently linked to the nucleic acid encoding a peptide ofthe present invention. Such tag peptides are well known in the art andinclude, for instance, green fluorescent protein (GFP), myc,myc-pyruvate kinase (myc-PK), His₆, maltose binding protein (MBP), aninfluenza virus hemagglutinin tag polypeptide, a flag tag polypeptide(FLAG), and a glutathione-S-transferase (GST) tag polypeptide. However,the invention should in no way be construed to be limited to the nucleicacids encoding the above-listed tag peptides. Rather, any nucleic acidsequence encoding a peptide which may function in a manner substantiallysimilar to these tag peptides should be construed to be included in thepresent invention.

[1122] The nucleic acid comprising a nucleic acid encoding a tag peptidecan be used to localize a peptide of the present invention within acell, a tissue, and/or a whole organism (e.g., a mammalian embryo),detect a peptide of the present invention secreted from a cell, and tostudy the role(s) of the peptide in a cell. Further, addition of a tagpeptide facilitates isolation and purification of the “tagged” peptidesuch that the peptides of the invention can be produced and purifiedreadily.

[1123] The invention includes the following preferred isolated peptides:G-CSF, IFN-alpha, IFN-beta, Factor VII, Factor IX, FSH, EPO, GM-CSF,IFN-gamma, A-1-PI, glucocerebrosidase, TPA, IL-2, Factor VIII, chimericTNFR, urokinase, chimeric anti-glycoprotein IIb/IIa, chimeric anti-HER2,chimeric anti-RSV, chimeric anti-CD20, DNase, chimeric anti-TNF, humaninsulin, HBsAg, HGH, alpha-galactosidase A, α-Iduronidase, antithrombinIII, hCG, and interferon omega, and the like.

[1124] The present invention should also be construed to encompass“derivatives,” “mutants”, and “variants” of the peptides of theinvention (or of the DNA encoding the same) which derivatives, mutants,and variants are peptides which are altered in one or more amino acids(or, when referring to the nucleotide sequence encoding the same, arealtered in one or more base pairs) such that the resulting peptide (orDNA) is not identical to the sequences recited herein, but has the samebiological property as the peptides disclosed herein, in that thepeptide has biological/biochemical properties of G-CSF, IFN-alpha,IFN-beta, Factor VII, Factor IX, FSH, EPO, GM-CSF, IFN-gamma, A-1-PI,glucocerebrosidase, TPA, IL-2, Factor VIII, chimeric TNFR, urokinase,chimeric anti-glycoprotein IIb/IIIa, chimeric anti-HER2, chimericanti-RSV, chimeric anti-CD20, DNase, chimeric anti-TNF, human insulin,HBsAg, and HGH.

[1125] Further included are fragments of peptides that retain thedesired biological activity of the peptide irrespective of the length ofthe peptide. It is well within the skill of the artisan to isolatesmaller than full length forms of any of the peptides useful in theinvention, and to determine, using the assays provided herein, whichisolated fragments retain a desired biological activity and aretherefore useful peptides in the invention.

[1126] A biological property of a protein of the present inventionshould be construed to include, but not be limited to include theability of the peptide to function in the biological assay andenvironments described herein, such as reduction of inflammation,elicitation of an immune response, blood-clotting, increasedhematopoietic output, protease inhibition, immune system modulation,binding an antigen, growth, alleviation of treatment of a disease, DNAcleavage, and the like.

[1127] A. G-CSF

[1128] The present invention encompasses a method for the modificationof the glycan structure on G-CSF. G-CSF is well known in the art as acytokine produced by activated T-cells, macrophages, endothelial cells,and stromal fibroblasts. G-CSF primarily acts on the bone marrow toincrease the production of inflammatory leukocytes, and furtherfunctions as an endocrine hormone to initiate the replenishment ofneutrophils consumed during inflammatory functions. G-CSF also hasclinical applications in bone marrow replacement following chemotherapy.

[1129] A remodeled G-CSF peptide may be administered to a patientselected from the group consisting of a non-myeloid cancer patientreceiving myelosuppressive chemotherapy, a patient having Acute MyeloidLeukemia (AML) receiving induction or consolidation chemotherapy, anon-myeloid cancer patient receiving a bone marrow transplant, a patientundergoing peripheral blood progenitor cell collection, a patient havingsevere chronic neutropenia, and a patient having persistent neutropeniaand also having advanced HIV infection. Preferably, the patient is ahuman patient.

[1130] While G-CSF has been shown to be an important and useful compoundfor therapeutic applications in mammals, especially humans, presentmethods for the production of G-CSF from recombinant cells results in aproduct having a relatively short biological life, an inaccurateglycosylation pattern that could potentially lead to immunogenicity,loss of function, and an increased need for both larger and morefrequent doses in order to achieve the same effect, and the like.

[1131] G-CSF has been isolated and cloned, the nucleic acid and aminoacid sequences of which are presented as SEQ ID NO:1 and SEQ ID NO:2,respectively (FIGS. 58A and 58B, respectively). The present inventionencompasses a method for modifying G-CSF, particularly as it relates tothe ability of G-CSF to function as a potent and functional biologicalmolecule. The skilled artisan, when equipped with the present disclosureand the teachings herein, will readily understand that the presentinvention provides compositions and methods for the modification ofG-CSF.

[1132] The present invention further encompasses G-CSF variants, as wellknown in the art. As an example, but in no way meant to be limiting tothe present invention, a G-CSF variant has been described in U.S. Pat.No. 6,166,183, in which a G-CSF comprising the natural complement oflysine residues and further linked to one or two polyethylene glycolmolecules is described. Additionally, U.S. Pat. Nos. 6,004,548,5,580,755, 5,582,823, and 5,676,941 describe a G-CSF variant in whichone or more of the cysteine residues at position 17, 36, 42, 64, and 74are replaced by alanine or alternatively serine. U.S. Pat. No. 5,416,195describes a G-CSF molecule in which the cysteine at position 17, theaspartic acid at position 27, and the serines at positions 65 and 66 aresubstituted with serine, serine, proline, and proline, respectively.Other variants are well known in the art, and are described in, forexample, U.S. Pat. No. 5,399,345.

[1133] The expression and activity of a modified G-CSF molecule of thepresent invention can be assayed using methods well known in the art,and as described in, for example, U.S. Pat. No. 4,810,643. As anexample, activity can be measured using radio-labeled thymidine uptakeassays. Briefly, human bone marrow from healthy donors is subjected to adensity cut with Ficoll-Hypaque (1.077 g/ml, Pharmacia, Piscataway,N.J.) and low density cells are suspended in Iscove's medium (GIBCO, LaJolla, Calif.) containing 10% fetal bovine serum, glutamine andantibiotics. About 2×10⁴ human bone marrow cells are incubated witheither control medium or the G-CSF or the present invention in 96-wellflat bottom plates at about 37° C. in 5% CO₂ in air for about 2 days.Cultures are then pulsed for about 4 hours with 0.5 μCi/well of³H-thymidine (New England Nuclear, Boston, Mass.) and uptake is measuredas described in, for example, Ventua, et al.(1983, Blood 61:781). Anincrease in ³H-thymidine incorporation into human bone marrow cells ascompared to bone marrow cells treated with a control compound is anindication of a active and viable G-CSF compound.

[1134] B. IFN Alpha, IFN Beta and IFN Omega

[1135] The present invention further encompasses a method for theremodeling and modification of IFN alpha, IFN beta and IFN omega. IFNalpha is part of a family of approximately twenty peptides ofapproximately 18 kDa in weight. IFN omega is very similar in structureand function to IFN alpha. IFN omega is useful for treatment ofhepatitis C virus infection when an immune response to IFN alpha ismounted in the host rendering that treatment ineffective. Antibodiesraised against IFN alpha do not cross-react with IFN omega. Thus,treatment of hepatitis C may continue using IFN omega when IFN alphatherapy is no longer possible.

[1136] IFN alpha, omega, and IFN beta, collectively known as the Type Iinterferons, bind to the same cellular receptor and elicit similarresponses. Type I IFNs inhibit viral replication, increase the lyticpotential of NK cells, modulate MHC molecule expression, and inhibitcellular proliferation, among other things. Type I IFN has been used asa therapy for viral infections, particularly hepatitis viruses, and as atherapy for multiple sclerosis.

[1137] Current compositions of Type I IFN are, as described above,useful compounds for both the modulation of aberrant immunologicalresponses and as a therapy for a variety of diseases. However, they arehampered by decreased potency and function, and a limited half-life inthe body as compared to natural cytokines comprising the naturalcomplement of glycosylation.

[1138] A remodeled interferon-alpha peptide may be administered to apatient selected from the group consisting of a patient having hairycell leukemia, a patient having malignant melanoma, a patient havingfollicular lymphoma, a patient having condylomata acuminata, a patienthaving AIDS-related Kaposi's sarcoma, a patient having Hepatitis C, apatient having Hepatitis B, a patient having a human papilloma virusinfection, a patient having Chronic Myeloid Leukemia (CML), a patienthaving chronic phase Philadelphia chromosome (Ph) positive ChronicMyelogenous Leukemia, a patient having non-Hodgkin's lymphoma (NHL), apatient having lymphoma, a patient having bladder cancer, and a patienthaving renal cancer. Preferably, the patient is a human patient.

[1139] A remodeled interferon-beta peptide may be administered to apatient selected from the group consisting of a patient having multiplesclerosis (MS), a patient having Hepatitis B, a patient having HepatitisC, a patient having human papilloma virus infection, a patient havingbreast cancer, a patient having brain cancer, a patient havingcolorectal cancer, a patient having pulmonary fibrosis, and a patienthaving rheumatoid arthritis. Preferably, the patient is a human patient.

[1140] A remodeled interferon-omega peptide may be administered to apatient selected from the group consisting of a patient having hairycell leukemia, a patient having malignant melanoma, a patient havingfollicular lymphoma, a patient having condylomata acuminata, a patienthaving AIDS-related Kaposi's sarcoma, a patient having Hepatitis C, apatient having Hepatitis B, a patient having a human papilloma virusinfection, a patient having Chronic Myeloid Leukemia (CML), a patienthaving chronic phase Philadelphia chromosome (Ph) positive ChronicMyelogenous Leukemia, a patient having non-Hodgkin's lymphoma (NHL), apatient having lymphoma, a patient having bladder cancer, and a patienthaving renal cancer. Preferably, the patient is a human patient.

[1141] The prototype nucleotide and amino acid sequence for IFN alpha isset forth herein as SEQ ID NO:3 and SEQ ID NO:4, respectively (FIGS. 59Aand 59B, respectively). The prototype nucleotide and amino acid sequencefor IFN omega is set forth herein as SEQ ID NO:74 and SEQ ID NO:75,respectively (FIGS. 84A and 84B, respectively). IFN beta comprises asingle gene product of approximately 20 kDa, the nucleic acid and aminoacid sequence of which are presented herein as SEQ ID NO:5 and SEQ IDNO:6 (FIGS. 60A and 60B, respectively). The present invention is notlimited to the nucleotide and amino acid sequences herein. One of skillin the art will readily appreciate that many variants of IFN alpha existboth naturally and as engineered derivatives. Similarly, IFN beta hasbeen modified in attempts to achieve a more beneficial therapeuticprofile. Examples of modified Type I IFNs are well known in the art (seeTable 9), and are described in, for example U.S. Pat. No. 6,323,006, inwhich cysteine-60 is substituted for tyrosine, U.S. Pat. Nos. 4,737,462,4,588,585, 5,545,723, and 6,127,332 where an IFN beta with asubstitution of a variety of amino acids is described. Additionally,U.S. Pat. Nos. 4,966,843, 5,376,567, 5,795,779 describe IFN alpha-61 andIFN-alpha-76. U.S. Pat. Nos. 4,748,233 and 4,695,543 describe IFN alphagx-1, whereas U.S. Pat. No. 4,975,276 describes IFN alpha-54. Inaddition, U.S. Pat. Nos. 4,695,623, 4,897,471, 5,661,009, and 5,541,293all describe a consensus IFN alpha sequence to represent all variantsknown at the date of filing. While this list of Type I IFNs and variantsthereof is in no way meant to be exhaustive, one of skill in the artwill readily understand that the present invention encompasses IFN betaand IFN alpha molecules, derivatives, and variants known or to bediscovered in the future. TABLE 9 Interferon-α Isoforms. α type AAcharacteristic 1a A¹¹⁴ 1b V¹¹⁴ 2a K²³-H³⁴ 2b R²³-H³⁴ 2c R²³-R³⁴ 4aA⁵¹-E¹¹⁴ 4b T⁵¹-V¹¹⁴ 7a M¹³²-K¹⁵⁹-G¹⁶¹ 7b M¹³²-Q¹⁵⁹-R¹⁶¹ 7cT¹³²-K¹⁵⁹-G¹⁶¹ 8a V⁹⁸-L⁹⁹-C¹⁰⁰-D¹⁰¹-R¹⁶¹ 8b S⁹⁸-C⁹⁹-V¹⁰⁰-M¹⁰¹-R¹⁶¹ 8cS⁹⁸-C⁹⁹-V¹⁰⁰-M¹⁰¹-D¹⁶¹Δ(162-166) 10a S⁸-L⁸⁹ 10b T⁸-I⁸⁹ 14aF¹⁵²-Q¹⁵⁹-R¹⁶¹ 14b F¹⁵²-K¹⁵⁹-G¹⁶¹ 14c L¹⁵²-Q¹⁵⁹-R¹⁶¹ 17a P³⁴-S⁵⁵-I¹⁶¹17b H³⁴-S⁵⁵-I¹⁶¹ 17c H³⁴-S⁵⁵-R¹⁶¹ 17d H³⁴-P⁵⁵-R¹⁶¹ 21a M⁹⁶ 21b L⁹⁶

[1142] Methods of expressing IFN in recombinant cells are well known inthe art, and is easily accomplished using techniques described in, forexample U.S. Pat. No. 4,966,843, and in Sambrook et al. (2001, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NewYork) and Ausubel et al. (1997, Current Protocols in Molecular Biology,Green & Wiley, New York). Assays to determine the biological activity ofa Type I IFN modified by the present invention will be well known to theskilled artisan. For example, the assay described in Rubinstein et al.,(1981, Journal of Virology 37:755-758) is commonly used to determine theeffect of an Type I IFN by measuring the cytopathic effects of viralinfection on a population of cells. This method is only one of manyknown in the art for assaying the biological function of a Type IFN.

[1143] C. Factor VIIa

[1144] The present invention further encompasses a method for theremodeling and modification of Factor VII. The blood coagulation pathwayis a complex reaction comprising many events. An intermediate event inthis pathway is Factor VII, a proenzyme that participates in theextrinsic pathway of blood coagulation by converting (upon itsactivation to Factor VIIa) Factor X to Xa in the presence of tissuefactor and calcium ions. Factor Xa in turn then converts prothrombin tothrombin in the presence of Factor Va, calcium ions and phospholipid.The activation of Factor X to Factor Xa is an event shared by both theintrinsic and extrinsic blood coagulation pathways, and therefore,Factor VIIa can be used for the treatment of patients with deficienciesor inhibitors of Factor VIII. There is also evidence to suggest thatFactor VIIa may participate in the intrinsic pathway as well thereforeincreasing the prominence and importance of the role of Factor VII inblood coagulation.

[1145] Factor VII is a single-chain glycoprotein with a molecular weightof approximately 50 kDa. In this form, the factor circulates in theblood as an inactive zymogen. Activation of Factor VII to VIIa may becatalyzed by several different plasma proteases, such as Factor XIIa.Activation of Factor VII results in the formation of a heavy chain and alight chain held together by at least one disulfide bond. Further,modified Factor VII molecules that cannot be converted to Factor VIIahave been described, and are useful as anti-coagulation remedies, suchas in the case of blood clots, thrombosis, and the like. Given theimportance of Factor VII in the blood coagulation pathway, and its useas a treatment for both increased and decreased levels of coagulation,it follows that a molecule that has a longer biological half-life,increased potency, and in general, a therapeutic profile more similar towild-type Factor VII as it is synthesized and secreted in the healthyhuman would be beneficial and useful as a treatment for bloodcoagulation disorders.

[1146] A remodeled Factor VII peptide may be administered to a patientselected from the group consisting of a hemophiliac patient having ableeding episode, a patient having Hemophilia A, a patient withHemophilia B, a patient having Hemophilia A, wherein the patient alsohas antibodies to Factor VIII, a patient having Hemophilia B, whereinthe patient also has antibodies to Factor IX, a patient having livercirrhosis, a cirrhotic patient having an orthotopic liver transplant, acirrhotic patient having upper gastrointestinal bleeding, a patienthaving a bone marrow transplant, and a patient having a liver resection.Preferably, the patient is a human patient.

[1147] Factor VII has been cloned and sequenced, and the nucleic acidand amino acid sequences are presented herein as SEQ ID NO:7 and SEQ IDNO:8 (FIGS. 61A and 61B, respectively). The present invention should inno way be construed as limited to the Factor VII nucleic acid and aminoacid sequences set forth herein. Variants of Factor VII are describedin, for example, U.S. Pat. Nos. 4,784,950 and 5,580,560, in whichlysine-38, lysine-32, arginine-290, arginine-341, isoleucine-42,tyrosine-278, and tyrosine-332 is replaced by a variety of amino acids.Further, U.S. Pat. Nos. 5,861,374, 6,039,944, 5,833,982, 5,788,965,6,183,743, 5,997,864, and 5,817,788 describe Factor VII variants thatare not cleaved to form Factor VIIa. The skilled artisan will recognizethat the blood coagulation pathway and the role of Factor VII thereinare well known, and therefore many variants, both naturally occurringand engineered, as described above, are included in the presentinvention.

[1148] Methods for the expression and to determine the activity ofFactor VII are well known in the art, and are described in, for example,U.S. Pat. No. 4,784,950. Briefly, expression of Factor VII, or variantsthereof, can be accomplished in a variety of both prokaryotic andeukaryotic systems, including E. coli, CHO cells, BHK cells, insectcells using a baculovirus expression system, all of which are well knownin the art.

[1149] Assays for the activity of a modified Factor VII preparedaccording to the methods of the present invention can be accomplishedusing methods well known in the art. As a non-limiting example, Quick etal. (Hemorragic Disease and Thrombosis, 2nd ed., Leat Febiger,Philadelphia, 1966), describes a one-stage clotting assay useful fordetermining the biological activity of a Factor VII molecule preparedaccording to the methods of the present invention.

[1150] D. Factor IX

[1151] The present invention further encompasses a method for remodelingand/or modifying Factor IX. As described above, Factor IX is vital inthe blood coagulation cascade. A deficiency of Factor IX in the bodycharacterizes a type of hemophilia (type B). Treatment of this diseaseis usually limited to intravenous tranfusion of human plasma proteinconcentrates of Factor IX. However, in addition to the practicaldisadvantages of time and expense, transfusion of blood concentratesinvolves the risk of transmission of viral hepatitis, acquired immunedeficiency syndrome or thromboembolic diseases to the recipient.

[1152] While Factor IX has demonstrated itself as an important anduseful compound for therapeutic applications, present methods for theproduction of Factor IX from recombinant cells (U.S. Pat. No. 4,770,999)results in a product with a rather short biological life, an inaccurateglycosylation pattern that could potentially lead to immunogenicity,loss of function, an increased need for both larger and more frequentdoses in order to achieve the same effect, and the like.

[1153] A remodeled Factor IX peptide may be administered to a patientselected from the group consisting of a hemophiliac patient having ableeding episode and also having Hemophilia B, a patient havingHemophilia B, a patient having Hemophilia B and having antibodies toFactor IX, a patient having liver cirrhosis, a cirrhotic patient havingan orthotopic liver transplant, a cirrhotic patient having uppergastrointestinal bleeding, a patient having a bone marrow transplant,and a patient having a liver resection. A remodeled Factor IX peptidemay also be administered to control and/or prevent hemorrhagic episodesin a patient having Hemophilia B, congenital Factor IX deficiency, orChristmas disease. A remodeled Factor IX peptide may also beadministered to a patient to control and/or prevent hemorrhagic episodesin the patient during surgery. Preferably, the patient is a humanpatient.

[1154] The nucleic and amino acid sequences of Factor IX is set forthherein as SEQ ID NO:9 and SEQ ID NO:10 (FIGS. 62A and 62B,respectively). The present invention is in no way limited to thesequences set forth herein. Factor IX variants are well known in theart, as described in, for example, U.S. Pat. Nos. 4,770,999, 5,521,070in which a tyrosine is replaced by an alanine in the first position,U.S. Pat. No. 6,037,452, in which Factor XI is linked to an alkyleneoxide group, and U.S. Pat. No. 6,046,380, in which the DNA encodingFactor IX is modified in at least one splice site. As demonstratedherein, variants of Factor IX are well known in the art, and the presentdisclosure encompasses those variants known or to be developed ordiscovered in the future.

[1155] Methods for determining the activity of a modified Factor IXprepared according to the methods of the present invention can becarried out using the methods described above, or additionally, usingmethods well known in the art, such as a one stage activated partialthromboplastin time assay as described in, for example, Biggs (1972,Human Blood Coagulation Haemostasis and Thrombosis (Ed. 1), Oxford,Blackwell, Scientific, pg. 614). Briefly, to assay the biologicalactivity of a Factor IX molecule developed according to the methods ofthe present invention, the assay can be performed with equal volumes ofactivated partial thromboplastin reagent, Factor IX deficient plasmaisolated from a patient with hemophilia B using sterile phlebotomytechniques well known in the art, and normal pooled plasma as standard,or the sample. In this assay, one unit of activity is defined as thatamount present in one milliliter of normal pooled plasma. Further, anassay for biological activity based on the ability of Factor IX toreduce the clotting time of plasma from Factor IX-deficient patients tonormal can be performed as described in, for example, Proctor andRapaport (1961, Amer. J. Clin. Path. 36: 212).

[1156] E. FSH

[1157] The present invention further includes a method for remodelingand/or modifying FSH. Human reproductive function is controlled in partby a family of heterodimeric human glycoprotein hormones which have acommon 92 amino acid glycoprotein alpha subunit, but differ in theirhormone-specific beta subunits. The family includes follicle-stimulatinghormone (FSH), luteinizing hormone (LH), thyrotropin orthyroid-stimulating hormone (TSH), and human chorionic gonadotropin(hCG). Human FSH and LH are used therapeutically to regulate variousaspects of metabolism pertinent to reproduction in the human female. Forexample, FSH partially purified from urine is used clinically tostimulate follicular maturation in anovulatory women with anovulatorysyndrome or luteal phase deficiency. Luteinizing hormone (LH) and FSHare used in combination to stimulate the development of ovarianfollicles for in vitro fertilization. The role of FSH in thereproductive cycle is sufficiently well-known to permit therapeutic use,but difficulties have been encountered due, in part, to theheterogeneity and impurity of the preparation from native sources. Thisheterogeneity is due to variations in glycosylation pattern.

[1158] FSH is a valuable tool in both in vitro fertilization andstimulation of fertilization in vivo, but as stated above, its clinicalefficacy has been hampered by inconsistency in glycosylation of theprotein. It therefore seems apparent that a method for remodeling FSHwill be of great benefit to the reproductive sciences.

[1159] A remodeled FSH peptide may be administered to a patient selectedfrom the group consisting of a patient undergoing intrauterineinsemination (IUI), a patient undergoing in vitro fertilization (IVF),and an infertile patient. A remodeled FSH peptide may also beadministered to induce or increase ovulation in a patient, to stimulatedevelopment of an ovarian follicle in a patient, to induce gametogenicfollicle growth in a patient, to stimulate, induce or increase follicledevelopment and subsequent ovulation in a patient, or to treatinfertility in a patient. Preferably, the patient is a human femalepatient. A remodeled FSH peptide may also be administered to a patienthaving a pituitary deficiency or to a patient during puberty. Preferablythis patient is a human male patient.

[1160] FSH has been cloned and sequenced, the nucleic and amino acidsequences of which are presented herein as SEQ ID NO:11, SEQ ID NO: 12,respectively (alpha subunit) and SEQ ID NO:13 and SEQ ID NO:14,respectively (beta subunit) (FIGS. 63A, 63B, 63C and 63D, respectively).The skilled artisan will readily appreciate that the present inventionis not limited to the sequences depicted herein, as variants of FSH arewell known in the art. As a non-limiting example, U.S. Pat. No.5,639,640 describes the beta subunit comprising two different amino acidsequences and U.S. Pat. No. 5,338,835 describes a beta subunitcomprising an additional amino acid sequence of approximatelytwenty-seven amino acids derived from the beta subunit of humanchorionic gonadotropin. Therefore, the present invention comprises FSHvariants, both natural and engineered by the human hand, all well knownin the art.

[1161] Methods to express FSH in cells, both prokaryotic and eukaryotic,are well known in the art and abundantly described in the literature(U.S. Pat. Nos. 4,840,896, 4,923,805, 5,156,957). Further, methods forevaluating the biological activity of a remodeled FSH molecule of thepresent invention are well known in the art, and are described in, forexample, U.S. Pat. No. 4,589,402, in which methods for determining theeffect of FSH on fertility, egg production, and pregnancy rates isdescribed in both non-human primates and human subjects.

[1162] F. EPO

[1163] The present invention further comprises a method of remodelingand/or modifying EPO. EPO is an acidic glycoprotein of approximately 34kDa and may occur in three natural forms: alpha, beta, and asialo. Thealpha and beta forms differ slightly in carbohydrate components but havethe same potency, biological activity and molecular weight. The asialoform is an alpha or beta form with the terminal sialic acid removed. EPOis present in very low concentrations in plasma when the body is in ahealthy state wherein tissues receive sufficient oxygenation from theexisting number of erythrocytes. This normal concentration is enough tostimulate replacement of red blood cells which are lost normally throughaging. The amount of erythropoietin in the circulation is increasedunder conditions of hypoxia when oxygen transport by blood cells in thecirculation is reduced. Hypoxia may be caused by loss of large amountsof blood through hemorrhage, destruction of red blood cells byover-exposure to radiation, reduction in oxygen intake due to highaltitudes or prolonged unconsciousness, or various forms of anemia.Therefore EPO is a useful compound for replenishing red blood cellsafter radiation therapy, anemia, and other life-threatening conditions.

[1164] A remodeled EPO peptide may be administered to a patient selectedfrom the group consisting of a patient having anemia, an anemic patienthaving chronic renal insufficiency, an anemic patient having end stagerenal disease, an anemic patient undergoing dialysis, an anemic patienthaving chronic renal failure, an anemic Zidovudine-treated HIV infectedpatient, an anemic patient having non-myeloid cancer and undergoingchemotherapy, and an anemic patient scheduled to undergo non-cardiac,non-vascular surgery. A remodeled EPO peptide may also be administeredto a patient undergoing surgery to reduce the need for an allogenicblood transfusion. A remodeled EPO peptide may also be administered to apatient at increased risk for a perioperative blood transfusion withsignificant anticipated blood loss. Preferably, the patient is a humanpatient.

[1165] In light of the importance of EPO in aiding in the recovery froma variety of diseases and disorders, the present invention is useful forthe production of EPO with a natural, and therefore more effectivesaccharide component. EPO, as it is currently synthesized, lacks thefull glycosylation complement, and must therefore be administered morefrequently and in higher doses due to its short life in the body. Theinvention also provides for the production of PEGylated EPO moleculeswith greatly improved half-life compared with what might be achieved bymaximizing desirable glycoforms.

[1166] EPO has been cloned and sequenced, and the nucleotide and aminoacid sequences are present herein as SEQ ID NO:15 and SEQ ID NO:16,respectively (FIGS. 64A and 64B, respectively). It will be readilyunderstood by one of skill in the art that the sequences set forthherein are only an example of the sequences encoding and comprising EPO.As an example, U.S. Pat. No. 6,187,564 describes a fusion proteincomprising the amino acid sequence of two or more EPO peptides, U.S.Pat. Nos. 6,048,971 and 5,614,184 describe mutant EPO molecules havingamino acid substitutions at positions 101, 103, 104, and 108. U.S. Pat.No. 5,106,954 describes a truncated EPO molecule, and U.S. Pat. No.5,888,772 describes an EPO analog with substitutions at position 33,139, and 166. Therefore, the skilled artisan will realize that thepresent invention encompasses EPO and EPO derivatives and variants asare well documented in the literature and art as a whole.

[1167] Additionally, methods of expressing EPO in a cell are well knownin the art. As exemplified in U.S. Pat. Nos. 4,703,008, 5,688,679, and6,376,218, among others, EPO can be expressed in prokaryotic andeukaryotic expression systems. Methods for assaying the biologicalactivity of EPO are equally well known in the art. As an example, theKrystal assay (Krystal, 1983, Exp. Hematol. 11:649-660) can be employedto determine the activity of EPO prepared according to the methods ofthe present invention. Briefly, the assay measures the effect oferythropoietin on intact mouse spleen cells. Mice are treated withphenylhydrazine to stimulate production of erythropoietin-responsive redblood cell progenitor cells. After treatment, the spleens are removed,intact spleen cells are isolated and incubated with various amounts ofwild-type erythropoietin or the erythropoietin proteins describedherein. After an overnight incubation, ³H-thymidine is added and itsincorporation into cellular DNA is measured. The amount of ³H-thymidineincorporation is indicative of erythropoietin-stimulated production ofred blood cells via interaction of erythropoietin with its cellularreceptor. The concentration of the erythropoietin protein of the presentinvention, as well as the concentration of wild-type erythropoietin, isquantified by competitive radioimmunoassay methods well known in theart. Specific activities are calculated as international units measuredin the Krystal assay divided by micrograms as measured asimmunoprecipitable protein by radioimmunoassay.

[1168] Several different mutated EPO's with different glycosylationpatterns have been reported. Many have improved stimulation ofreticulocytosis activity without effecting the half-life of the peptidein the blood stream of the animal. It is contemplated that mutated EPOpeptides can be used in place of the native EPO peptides in any of theglycan remodeling, glycoPEGylation and/or glycoconjugation embodimentsdescribed herein. Preferred mutations of EPO are listed in the followingtable, but not limited to those listed in the table (see, for example,Chern et al., 1991, Eur. J. Biochem. 202:225-229; Grodberg et al., 1993,Eur. J. Biochem. 218:597-601; Burns et al., 2002, Blood 99:4400-4405;U.S. Pat. No. 5,614,184; GenBank Accession No. AAN76993; O'Connell etal., 1992, J. Biol. Chem. 267:25010-25018; Elliott et al., 1984, Proc.Natl. Acad. Sci. U.S.A. 81:2708-2712; Biossel et al., 1993, J. Biol.Chem. 268:15983-15993). The most preferred mutations of EPO are Arg¹³⁹to Ala¹³⁹, Arg¹⁴³ to Ala¹⁴³ and Lys¹⁵⁴ to Ala¹⁵⁴. The preferred nativeEPO from which to make these mutants is the 165 aa form, which isdepicted in FIG. 65; however other native forms of EPO may also be used.Finally, the mutations described in Table 10 may be combined with eachother and with other mutations to make EPO peptides that are useful inthe present invention. TABLE 10 Mutations of EPO. Mutation CitationNotes Arg¹³⁹ to Ala¹³⁹ J. Biol. Chem. 269: 22839 Increased activity in(1994) bioassays of 120% to 150%. Arg¹⁴³ to Ala¹⁴³ Increased activity inbioassays than native EPO. Lys¹⁵⁴ to Ala¹⁵⁴ J. Biol. Chem. 269: 22839Increased activity in (1994) bioassays of 120% to 150%. Ser¹²⁶ to Met¹²⁶Met⁵⁴ to Leu⁵⁴ U.S. Pat. No. 4,385,260 Met⁵⁴ to Leu⁵⁴ U.S. Pat. No.4,385,260 Asn³⁸ to Gln³⁸ Δ1-30 Funakoshi et al., 1993, Mutant isolatedfrom hepatocellular Ser¹³¹Leu¹³² to Biochem. Biophys. Res. carcinoma.Asn¹³¹Phe¹³² Commun. 195: 717-722. Pro¹⁴⁹ to Gln¹⁴⁹ Genbank AccessionNo. AAD13964. Gly¹⁰¹ to Ala¹⁰¹ U.S. Pat. No. 5,615,184 Increasedactivity in J. Biol. Chem. 269: 22839 bioassays of (1994) 120% to 150%.Ser¹⁴⁷ to Ala¹⁴⁷ Wen et al., 1994, J. Biol. Mutation results in and/orChem. 269: 22839-22846. increased bioactivity. Ile¹⁴⁶ to Ala¹⁴⁶ Ser¹²⁶to Thr¹²⁶ J. Biol. Chem. 267: 25010 (1992)

[1169] G. GM-CSF

[1170] The present invention encompasses a method for the modificationof GM-CSF. GM-CSF is well known in the art as a cytokine produced byactivated T-cells, macrophages, endothelial cells, and stromalfibroblasts. GM-CSF primarily acts on the bone marrow to increase theproduction of inflammatory leukocytes, and further functions as anendocrine hormone to initiate the replenishment of neutrophils consumedduring inflammatory functions. Further GM-CSF is a macrophage-activatingfactor and promotes the differentiation of Lagerhans cells intodendritic cells. Like G-CSF, GM-CSF also has clinical applications inbone marrow replacement following chemotherapy.

[1171] While G-CSF has demonstrated itself as an important and usefulcompound for therapeutic applications, present methods for theproduction of G-CSF from recombinant cells results in a product with arather short biological life, an inaccurate glycosylation pattern thatcould potentially lead to immunogenicity, loss of function, an increasedneed for both larger and more frequent doses in order to achieve thesame effect, and the like.

[1172] A remodeled GM-CSF peptide may be administered to a patientselected from the group consisting of a patient having Acute MyelogenousLeukemia (AML) or acute non-lymphocytic leukemia (ANLL), a patientundergoing leukapheresis to collect hematopoietic progenitor cells fromthe peripheral blood, a patient undergoing transplantation of autologousperipheral blood progenitor cells, a non-Hodgkin's lymphoma (NHL)patient undergoing an autologous bone marrow transplant, a Hodgkin'sdisease patient undergoing an autologous bone marrow transplant, and anacute lymphoblastic leukemia (ALL) patient undergoing an autologous bonemarrow transplant. A remodeled GM-CSF peptide may also be administeredto a patient to accelerate myeloid engraftment, to shorten time toneutrophil recovery following chemotherapy, to mobilize hematopoieticprogenitor cells into the peripheral blood for collection byleukapheresis, or to promote myeloid reconstitution after autologous orallogeneic bone marrow transplantation (BMT). A remodeled GM-CSF peptidemay also be administered to a patient in which bone marrowtransplantation has failed or in which myeloid engraftment is delayed.Preferably, the patient is a human patient.

[1173] GM-CSF has been isolated and cloned, the nucleic acid and aminoacid sequences of which are presented as SEQ ID NO: 17 and SEQ ID NO:18, respectively (FIGS. 66A and 66B, respectively). The presentinvention encompasses a method for modifying GM-CSF, particularly as itrelates to the ability of GM-CSF to function as a potent and functionalbiological molecule. The skilled artisan, when equipped with the presentdisclosure and the teachings herein, will readily understand that thepresent invention provides compositions and methods for the modificationof GM-CSF.

[1174] The present invention further encompasses GM-CSF variants, aswell known in the art. As an example, but in no way meant to be limitingto the present invention, a GM-CSF variant has been described in WO86/06358, where the protein is modified for an alternative quaternarystructure. Further, U.S. Pat. No. 6,287,557 describes a GM-CSF nucleicacid sequence ligated into the genome of a herpesvirus for gene therapyapplications. Additionally, European Patent Publication No. 0288809(corresponding to PCT Patent Publication No. WO 87/02060) reports afusion protein comprising IL-2 and GM-CSF. The IL-2 sequence can be ateither the N- or C-terminal end of the GM-CSF such that after acidcleavage of the fusion protein, GM-CSF having either N- or C-terminalsequence modifications can be generated. Therefore, GM-CSF derivatives,mutants, and variants are well known in the art, and are encompassedwithin the methods of the present invention.

[1175] The expression and activity of a modified GM-CSF molecule of thepresent invention can be assayed using methods well known in the art,and as described in, for example, U.S. Pat. No. 4,810,643. As anexample, activity can be measured using radio-labeled thymidine uptakeassays. Briefly, human bone marrow from healthy donors is subjected to adensity cut with Ficoll-Hypaque (1.077 g/ml, Pharmacia, Piscataway,N.J.) and low density cells are suspended in Iscove's medium (GIBCO, LaJolla, Calif.) containing 10% fetal bovine serum, glutamine andantibiotics. About 2×10⁴ human bone marrow cells are incubated witheither control medium or the GM-CSF or the present invention in 96-wellflat bottom plates at about 37° C. in 5% CO₂ in air for about 2 days.Cultures are then pulsed for about 4 hours with 0.5 μCi/well of³H-thymidine (New England Nuclear, Boston, Mass.) and uptake is measuredas described in, for example, Ventua, et al.(1983, Blood 61:781). Anincrease in ³H-thymidine incorporation into human bone marrow cells ascompared to bone marrow cells treated with a control compound is anindication of a active and viable GM-CSF compound.

[1176] H. IFN-gamma

[1177] It is an object of the present invention to encompass a method ofmodifying and/or remodeling IFN-gamma. IFN-gamma, otherwise known asType II interferon, in contrast to IFN alpha and IFN beta, is ahomodimeric glycoprotein comprising two subunits of about 21-24 kDa. Thesize variation is due to variable glycosylation patterns, usually notreplicated when reproduced recombinantly in various expression systemsknown in the art. IFN-gamma is a potent activator of macrophages,increases MHC class I molecule expression, and to a lesser extent, a MHCclass II molecule stimulatory agent. Further, IFN-gamma promotes T-celldifferentiation and isotype switching in B-cells. IFN-gamma is also welldocumented as a stimulator of neutrophils, NK cells, and antibodyresponses leading to phagocyte-mediated clearance. IFN-gamma has beenproposed as a treatment to be used in conjunction with infection byintracellular pathogens, such as tuberculosis and leishmania, and alsoas an anti-proliferative therapeutic, useful in conditions with abnormalcell proliferation as a hallmark, such as various cancers and otherneoplasias.

[1178] IFN-gamma has demonstrated potent immunological activity, but dueto variations in glycosylation from systems currently used to expressIFN-gamma, the potency, efficacy, biological half-life, and otherimportant factors of a therapeutic have been variable at best. Thepresent invention encompasses methods to correct this crucial defect.

[1179] A remodeled interferon-gamma peptide may be administered to apatient selected from the group consisting of a patient having chronicgranulomatous disease, a patient having malignant osteopetrosis, apatient having pulmonary fibrosis, a patient having tuberculosis, apatient having Cryptococcal meningitis, and a patient having pulmonaryMycobacterium avium complex (MAC) infection. Preferably, the patient isa human patient.

[1180] The nucleotide and amino acid sequences of IFN-gamma arepresented herein as SEQ ID NO:19 and SEQ ID NO:20, respectively (FIGS.67A and 67B, respectively). It will be readily understood that thesequences set forth herein are in no way limiting to the presentinvention. In contrast, variants, derivatives, and mutants of IFN-gammaare well known to the skilled artisan. As an example, U.S. Pat. No.6,083,724 describes a recombinant avian IFN-gamma and U.S. Pat. No.5,770,191 describes C-terminus variants of human IFN-gamma. In addition,U.S. Pat. No. 4,758,656 describes novel IFN-gamma derivatives, andmethods of synthesizing them in various expression systems. Therefore,the present invention is not limited to the sequences of IFN-gammadisclosed elsewhere herein, but encompasses all derivatives, variants,muteins, and the like well known in the art.

[1181] Expression systems for IFN-gamma are equally well known in theart, and include prokaryotic and eukaryotic systems, as well as plantand insect cell preparations, methods of which are known to the skilledartisan. As an example, U.S. Pat. No. 4,758,656 describes a system forexpressing IFN-gamma derivatives in E. coli, whereas U.S. Pat. No.4,889,803 describes an expression system employing Chinese hamster ovarycells and an SV40 promoter.

[1182] Assays for the biological activity of a remodeled IFN-gammaprepared according to the methods disclosed herein will be well known toone of skill in the art. Biological assays for IFN-gamma expression canbe found in, for example, U.S. Pat. No. 5,807,744. Briefly, IFN-gamma isadded to cultures of CD34⁺⁺CD38⁻ cells (100 cells per well) stimulatedby cytokine combinations to induce proliferation of CD34⁺⁺CD38⁻ cells,such as IL-3, c-kit ligand and either IL-1, IL-6 or G-CSF. Cellproliferation, and generation of secondary colony forming cells will beprofoundly inhibited in a dose dependent way, with near completeinhibition occurring at 5000 U/milliliter of IFN-gamma. As aconfirmatory test to the inhibitory effect of IFN-gamma, addition ofIFN-gamma antibodies can be performed as a control.

[1183] I. Alpha-Protease Inhibitor (α-Antitrypsin)

[1184] The present invention further includes a method for theremodeling of alpha-protease inhibitor (A-1-PI, α-1-antitrypsin orα-1-trypsin inhibitor), also known as alpha-antitrypsin. A-1-PI is aglycoprotein having molecular weight of 53 kDa. A-1-PI plays a role incontrolling tissue destruction by endogenous serine proteases, and isthe most pronounced serine protease inhibitor in blood plasma. Inparticular, A-1-PI inhibits various elastases including neutrophilelastase. Elastase is a protease which breaks down tissues, and can beparticularly problematic when its activity is unregulated in lungtissue. This protease functions by breaking down foreign proteins.However, when API is not present in sufficient quantities to regulateelastase activity, the elastase breaks down lung tissue. In time, thisimbalance results in chronic lung tissue damage and emphysema. In fact,a genetic deficiency of A-1-PI has been shown to be associated withpremature development of pulmonary emphysema. A-1-PI replenishment hasbeen successfully used for treatment of this form of emphysema. Further,a deficiency of A-1-PI may also contribute to the aggravation of otherdiseases such as cystic fibrosis and arthritis, where leukocytes move into the lungs or joints to fight infection.

[1185] Therefore, A-1-PI could conceivably be used to treat diseaseswhere an imbalance between inhibitor and protease(s), especiallyneutrophil elastase, is causing progression of a disease state.Antiviral activity has also been attributed to A-1-PI. In light of this,it logically follows that the present invention is useful for theproduction of A-1-PI that is safe, effective, and potent in the everchanging atmosphere of the lungs.

[1186] A remodeled A-1-PIpeptide may be administered to a patientselected from the group consisting of a patient having congenitalalpha-1-antitrypsin deficiency and emphysema, a patient having cysticfibrosis, and a patient having pulmonary fibrosis. Preferably, thepatient is a human patient.

[1187] A-1-PI has been cloned and sequenced, and is set forth in SEQ IDNO:21 and SEQ ID NO:22 (FIGS. 68A and 68B, respectively). As isunderstood by one of skill in the art, natural and engineered variantsof A-1-PI exist, and are encompassed in the present invention. As anexample, U.S. Pat. No. 5,723,316 describes A-1-PI derivatives havingamino acid substitutions at positions 356-361 and further comprises anN-terminal extension of approximately three amino acids. U.S. Pat. No.5,674,708 describes A-1-PI analogs with amino acid substitutions atposition 358 in the primary amino acid sequence. The skilled artisanwill readily realize that the present invention encompasses A-1-PIvariants, derivatives, and mutants known or to be discovered.

[1188] Methods for the expression and determination of activity of aremodeled A-1-PI produced according to the methods of the presentinvention are well known in the art, and are described in, for example,U.S. Pat. No. 5,674,708 and U.S. Pat. No. 5,723,316. Briefly, biologicalactivity can be determined using assays for antichymotrypsin activity bymeasuring the inhibition of the chymotrypsin-catalyzed hydrolysis ofsubstrate N-suc-Ala-Ala-Pro-Phe-p-nitroanilide (0.1 ml of a 10 mMsolution in 90% DMSO), as described in, for example, DelMar et al.(1979, Anal. Biochem. 99: 316). A typical chymotrypsin assay contains,in 1.0 milliliters: 100 mM Tris-C1 buffer, pH 8.3, 0.005% (v/v) TritonX-100, bovine pancreatic chymotrypsin (18 kmmol) and A-1-PI of thepresent invention. The assay mixture is pre-incubated at roomtemperature for 5 minutes, substrate (0.01 ml of a 10 mM solution in 90%DMSO) is added and remaining chymotrypsin activity is determined by therate of change in absorbance at 410 nm caused by the release ofp-nitroaniline. Measurements of optical absorbance are conducted at 25°C. using a spectrophotometer fitted with a temperature controlled samplecompartment.

[1189] J. Glucocerebrosidase

[1190] The invention described herein further includes a method for themodification of glucocerebrosidase. Glucocerebrosidase is a lysosomalglycoprotein enzyme which catalyzes the hydrolysis of the glycolipidglucocerebroside to glucose and ceramide. Variants of glucocerebrosidaseare sold commercially as Cerezyme™ and Ceredase™, and is an approvedtherapeutic for the treatment of Gaucher disease. Ceredase™ is aplacental derived form of glucocerebrosidase with complete N-linkedstructures. Cerezyme™ is a recombinant variant of glucocerebrosidasewhich is 497 amino acids in length and is expressed in CHO cells. The 4N-linked glycans of Cerezyme have been modified to terminate in thetrimannose core.

[1191] Glucocerebrosidase is presently produced in recombinant mammaliancell cultures, and therefore reflects the glycosylation patterns ofthose cells, usually rodent cells such as Chinese hamster ovary cells orbaby hamster kidney cells, which differ drastically from those of humanglycosylation patterns, leading to, among other things, immunogenicityand lack of potency.

[1192] A remodeled glucocerebrosidase peptide may be administered to apatient selected from the group consisting of a patient having alysosomal storage disease, a patient having a glucocerebrosidasedeficiency, and a patient having Gaucher disease. Preferably, thepatient is a human patient.

[1193] The nucleic acid and amino acid sequences of glucocerebrosidaseare set forth herein as SEQ ID NO:23 and 24 (FIGS. 69A and 69B,respectively). However, as will be appreciated by the skilled artisan,the sequences represented herein are prototypical sequences, and do notlimit the invention. In fact, variants of glucocerebrosidase are wellknown, and are described in, for example, U.S. Pat. No. 6,015,703describes enhanced production of glucocerebrosidase analogs and variantsthereof. Further, U.S. Pat. No. 6,087,131 describes the cloning andsequencing of yet another glucocerebrosidase variant. It is theintention of the present invention to encompass these and otherderivatives, variants, and mutants known or to be discovered in thefuture.

[1194] Methods for the expression of glucocerebrosidase are well knownin the art using standard techniques, and are described in detail in,for example, U.S. Pat. No. 6,015,703. Assays for the biological efficacyof a glucocerebrosidase molecule prepared according to the methods ofthe present invention are similarly well known in the art, and a mouseGaucher disease model for evaluation and use of a glucocerebrosidasetherapeutic is described in, for example, Marshall et al. (2002, Mol.Ther. 6:179).

[1195] K. TPA

[1196] The present invention further encompasses a method for theremodeling of tissue-type activator (TPA). TPA activates plasminogen toform plasmin which dissolves fibrin, the main component of the proteinsubstrate of the thrombus. TPA preparations were developed as athrombolytic agents having a very high selectivity toward the thrombusin the thrombolytic treatment for thrombosis which causes myocardialinfarction and cerebral infarction.

[1197] Further, various modified TPA's have been produced by geneticengineering for the purpose of obtaining higher affinity to fibrin andlonger half-life in blood than that of natural TPA. TPA's are proteinsthat are generally extremely difficult to solubilize in water. Inparticular, the modified TPA's are more difficult to solubilize in waterthan natural TPA, making very difficult the preparation of modifiedTPA's. Modified TPA's are thus difficult to dissolve in water at thetime of the administration to a patient. However, the modified TPA'shave various advantages, such as increased affinity for fibrin andlonger half-life in blood. It is the object of the present invention toincrease the solubility of modified TPA's.

[1198] A remodeled TPA peptide may be administered to a patient selectedfrom the group consisting of a patient suffering from an acutemyocardial infarction and a patient suffering from an acute ischemicstroke. A remodeled TPA peptide may also be administered to a patient toimprove ventricular function following an acute myocardial infarction,to reduce the incidence of congestive heart failure following an acutemyocardial infarction, or to reduce mortality associated with acutemyocardial infarction. A remodeled TPA peptide may also be administeredto a patient to improve neurological recovery following an acuteischemic stroke or to reduce the incidence of disability or paralysisfollowing an acute ischemic stroke. Preferably, the patient is a humanpatient.

[1199] The nucleic and amino acid sequences of TPA are set forth hereinas SEQ ID NO:25 and SEQ f) NO:26, respectively (FIGS. 70A and 70B,respectively). As described above, variants of TPA have been constructedand used in therapeutic applications. For example, U.S. Pat. No.5,770,425 described TPA variants in which some of all of the fibrindomain has been deleted. Further, U.S. Pat. No. 5,736,134 describes TPAin which modifications to the amino acid at position 276 are disclosed.The skilled artisan, when equipped with the present disclosure and theteachings herein, will readily realize that the present inventioncomprises the TPA sequences set forth herein, as well as those variantswell known to one versed in the literature.

[1200] The expression of TPA from a nucleic acid sequence encoding thesame is well known in the art, and is described, in detail, in, forexample, U.S. Pat. No. 5,753,486. Assays for determining the biologicalproperties of a TPA molecule prepared according to the methods of thepresent invention are similarly well known in the art. Briefly, a TPAmolecule synthesized as disclosed elsewhere herein can be assayed fortheir ability to lyse fibrin in the presence of saturatingconcentrations of plasminogen, according to the method of Carlsen et al.(1988, Anal. Biochem. 168: 428). The in vitro clot lysis assay measuresthe activity of tissue-type activators by turbidimetry using amicrocentrifugal analyzer. A mixture of thrombin and TPA is centrifugedinto a mixture of fibrinogen and plasminogen to initiate clot formationand subsequent clot dissolution. The resultant profile of absorbanceversus time is analyzed to determine the assay endpoint. Activities ofthe TPA variants are compared to a standard curve of TPA. The bufferused throughout the assay is 0.06M sodium phosphate, pH 7.4 containing0.01% (v/v) TWEEN 80 and 0.01% (w/v) sodium azide. Human thrombin is ata concentration of about 33 units/ml. Fibrinogen (at 2.0 mg/ml clottableprotein) is chilled on wet ice to precipitate fibronectin and thengravity filtered. Glu-plasminogen is at a concentration of 1 mg/ml. Theanalyzer chamber temperature is set at 37° C. The loader is set todispense 20 microliters of TPA (about 500 nanograms/milliliter to about1.5 micrograms per milliliter) as the sample for the standard curve, or20 microliters of variant TPAs at a concentration to cause lysis withinthe range of the standard curve. Twenty microliters of thrombin as thesecondary reagent, and 200 microliters of a 50:1 (v/v) fibrinogen:plasminogen mixture as the primary reagent. The absorbance/time programis used with a 5 min incubation time, 340-nanometer-filter and 90 secondinterval readings.

[1201] L. IL-2

[1202] The present invention further encompasses a method for theremodeling and modification of IL-2. IL-2 is the main growth factor of Tlymphocytes and increases the humoral and cellular immune responses bystimulating cytotoxic CD8 T cells and NK cells. IL-2 is thereforecrucial in the defense mechanisms against tumors and viral infections.IL-2 is also used in therapy against metastatic melanoma and renaladenocarcinoma, and has been used in clinical trials in many forms ofcancer. Further, IL-2 has also been used in HIV infected patients whereit leads to a significant increase in CD4 counts.

[1203] Given the success IL-2 has demonstrated in the management andtreatment of life-threatening diseases such as various cancers and AIDS,it follows that the methods of the present invention would be useful fordeveloping an IL-2 molecule that has a longer biological half-life,increased potency, and in general, a therapeutic profile more similar towild-type IL-2 as it is synthesized secreted in the healthy human.

[1204] A remodeled IL-2 peptide may be administered to a patientselected from the group consisting of a patient having metastatic renalcell carcinoma, a patient having metastatic melanoma, a patient havingovarian cancer, a patient having Acute Myelogenous Leukemia (AML), apatient having non-Hodgkin's lymphoma (NHL), a patient infected withHIV, and a patient infected with Hepatitis C. A remodeled IL-2 peptidemay also be useful for administeration to a patient as a cancer vaccineadjuvant. Preferably, the patient is a human patient.

[1205] IL-2 has been cloned and sequenced, and the nucleic acid andamino acid sequences are presented herein as SEQ ID NO:27 and SEQ IDNO:28 (FIGS. 71A and 71B, respectively). The present invention should inno way be construed as limited to the IL-2 nucleic acid and amino acidsequences set forth herein. Variants of IL-2 are described in, forexample, U.S. Pat. No. 6,348,193, in which the asparagine at position 88is substituted for arginine, and in U.S. Pat. No. 5,206,344, in which apolymer comprising IL-2 variants with various amino acid substitutionsis described. The present invention encompasses these IL-2 variants andothers well known in the art.

[1206] Methods for the expression and to determine the activity of IL-2are well known in the art, and are described in, for example, U.S. Pat.No. 5,417,970. Briefly, expression of IL-2, or variants thereof, can beaccomplished in a variety of both prokaryotic and eukaryotic systems,including E. coli, CHO cells, BHK cells, insect cells using abaculovirus expression system, all of which are well known in the art.

[1207] Assays for the activity of a modified IL-2 prepared according tothe methods of the present invention can proceed as follows. Peripheralblood lymphocytes can be separated from the erythrocytes andgranulocytes by centrifuging on a Ficoll-Hypaque (Pharmacia, Piscataway,N.J.) gradient by the method described in, for example, A. Boyum et al.(Methods in Enzymology, 1984, Vol. 108, page 88, Academic Press, Inc.).Lymphocytes are subsequently washed about three times in culture mediumconsisted of RPMI 1640 (Gibco-BRL, La Jolla, Calif.) plus 10% AB humanserum (CTS Purpan, Toulouse, France) inactivated by heat (1 hour at 56°C.), 2 mM sodium pyruvate, 5 mM HEPES, 4 mM L-glutamine, 100 U/mlpenicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B(complete medium). Adhesive cells (monocytes and macrophages) areeliminated by adhesion to plastic and the remainder of the cells aresuspended in complete medium at a concentration of about 5 to 10×10⁵cells per milliliter and seeded in culture flasks at a density of about1-2×10⁵ cells per square centimeter. Flasks are then incubated at 370 Cin a 5% CO₂ atmosphere for about 1 hour, after which the non-adhesivelymphocytes are recovered by aspiration after gentle agitation of theculture flasks.

[1208] Non-adhesive lymphocytes are washed once and cultivated at aconcentration of about 10⁵ cells per milliliter in complete medium inthe presence of the IL-2 of the present invention for about 48 hours inan incubator as described above. The cells are then washed once.

[1209] The cytotoxic activity of the cells is evaluated after about 4hours of contact with target cells of the human T lymphoid lineC8166-45/C63 (HT1 cells) resistant to NK cell cytotoxicity, as describedby Salahuddin et al. (1983, Virology 129: 51-64; 1984, Science: 223,703-707). 6×10⁵ HT1 cells are radio-tagged with about 200 μCi of ⁵¹Cr(sodium chromate, Amersham, Arlington Heights, Ill.) at 37° C. for about1 hour in complete medium without serum, and then washed several times.The target cells and effective cells are distributed in round-bottomedmicrotitration plates with varying ratios of effective cells to targetcells (50:1, 10:1, 1:1). The microtitration plates are centrifuged and,after incubation as described above, the supernatant from each well isrecovered and the radioactivity is measured using a gamma counter.Cytotoxicity is determined from the quantity of ⁵¹Cr released by deadtarget cells. Non-specific cytotoxicity is determined from the amount ofradioactivity spontaneously released from the target cells in theabsence of effective cells.

[1210] The present method is just one of many well known in the art formeasuring the cytotoxicity of effector cells, and is should not beconstrued as limiting to the present invention.

[1211] M. Factor VIII

[1212] The invention further encompasses a method for the remodeling andmodification of Factor VIII. As described earlier for Factor VII andFactor IX, Factor VIII is a critical component of the blood coagulationpathway. Human Factor VIII, (antihemophilic factor; FVIII:C) is a humanplasma protein consisting of 2 peptides (light chain molecular weight of80 kDa and heavy chain molecular weight variable from 90 to 220 kDa,depending on glycosylation state). It is an essential cofactor in thecoagulation pathway and is required for the conversion of Factor X intoits active form (Factor Xa). Factor VIII circulates in plasma as anon-covalent complex with von Willibrand Factor (aka FVIII:RP), a dimerof a 2050 aa peptide (See, U.S. Pat. No. 6,307,032). Bloodconcentrations of Factor VIII below 20% of normal cause a bleedingdisorder designated hemophilia A. Factor VIII blood levels less than 1%result in a severe bleeding disorder, with spontaneous joint bleedingbeing the most common symptom.

[1213] Similar to other blood coagulation factors, Factor VIII is atherapeutic with a great deal of potential for the treatment of variousbleeding disorders, such as hemophilia A and hemophilia B. Due to theglycosylation of the heavy chain, current methods for the preparation ofFactor VIII from recombinant cells results in a product that is not aseffective as natural Factor VIII. Purification methods from human plasmaresult in a crude composition that is less effective and more difficultto prepare than recombinant Factor VIII. The current invention seeks toimprove this situation.

[1214] A remodeled Factor VIII peptide may be administered to a patientselected from the group consisting of a patient having von Willebrand'sdisease, a patient having Hemophilia A, a patient having Factor VIII:Cdeficiency, a patient having fibrinogen deficiency, a patient havingFactor XIII deficiency, and a patient having acquired Factor VIIIinhibitors (acquired hemophilia). A remodeled Factor VIII peptide mayalso be administered to a patient to prevent, treat or control bleedingor hemorrhagic episodes. Preferably, the patient is a human patient.

[1215] The nucleic acid and amino acid sequences of Factor VIII arepresented herein as SEQ ID NO:29 and SEQ ID NO:30, respectively (FIGS.72A and 72B, respectively). The art is rife with variants of FactorVIII, as described in, for example, U.S. Pat. No. 5,668,108, in whichthe aspartic acid at position 1241 is replaced by a glutamic acid withthe accompanying nucleic acid changes as well. U.S. Pat. No. 5,149,637describes a Factor VIII variants comprising the C-terminal fraction,either glycosylated or unglycosylated, and U.S. Pat. No. 5,661,008describes a Factor VIII variant comprising amino acids 1-740 linked toamino acids 1649 to 2332 by at least 3 amino acid residues. Therefore,variants, derivatives, modifications and complexes of Factor VIII arewell known in the art, and are encompassed in the present invention.

[1216] Expression systems for the production of Factor VIII are wellknown in the art, and include prokaryotic and eukaryotic cells, asexemplified in U.S. Pat. Nos. 5,633,150, 5,804,420, and 5,422,250.

[1217] To determine the biological activity of a Factor VIII moleculesynthesized according the methods of the present invention, the skilledartisan will recognize that the assays described herein for theevaluation of Factor VII and Factor IX are applicable to Factor VIII.

[1218] N. Urokinase

[1219] The present invention also includes a method for the remodelingand/or modification of urokinase. Urokinase is a serine protease whichactivates plasminogen to plasmin. The protein is synthesized in avariety of tissues including endothelium and kidney, and is excreted intrace amounts into urine. Purified urokinase exists in two active forms,a high molecular weight form (HUK; approximately 50 kDa) and a lowmolecular weight form (LUK; approximately 30 kDa). LUK has been shown tobe derived from HUK by a proteolysis after lysine 135, releasing thefirst 135 amino acids from HUK. Conventional wisdom has held that HUK orLUK must be converted to proteolytically active forms by the proteolytichydrolysis of a single chain precursor, also termed prourokinase,between lysine 158 and isoleucine 159 to generate a two-chain activatedform (which continues to correspond to either HUK or LUK). Theproteolytically active urokinase species resulting from this hydrolyticclip contains two amino acid chains held together by a single disulfidebond. The two chains formed by the activation clip are termed the A orA₁ chains (HUK or LUK, respectively), and the B chain comprising theprotease domain of the molecule.

[1220] Urokinase has been shown to be an effective thrombolytic agent.However, since it is produced naturally in trace quantities the cost ofthe enzyme is high for an effective dosage. Urokinase has been producedin recombinant cell culture, and DNA encoding urokinase is knowntogether with suitable vectors and host microorganisms. Presentcompositions comprising urokinase and methods for producing urokinaserecombinantly are hampered by a product that has deficient glycosylationpatterns, and given the complex proteolytic cleavage events surroundingthe activation of urokinase, this aberrant glycosylation leads to a lesseffective and less potent product.

[1221] A remodeled urokinase peptide may be administered to a patientselected from the group consisting of a patient having an embolism, apatient having an acute massive pulmonary embolism, and a patient havingcoronary artery thrombosis. Preferably, the patient is a human patient.A remodeled urokinase peptide may also be used to restore patency to anintravenous catheter, including a central venous catheter obstructed byclotted blood or fibrin.

[1222] The sequence of the nucleotides encoding the primary amino acidchain of urokinase are depicted in SEQ ID NO:33 and SEQ ID NO:34 (FIGS.73A and 73B, respectively). Variants of urokinase are well known in theart, and therefore the present invention is not limited to the sequencesset forth herein. In fact, the skilled artisan will readily realize thaturokinase variants described in, for example U.S. Pat. Nos. 5,219,569,5,648,253, and 4,892,826, exist as functional moieties, and aretherefore encompassed in the present invention.

[1223] The expression and evaluation of a urokinase molecule preparedaccording to the methods of the present invention are similarly wellknown in the art. As a non-limiting example, the expression of urokinasein various systems is detailed in U.S. Pat. No. 5,219,569. An assay fordetermining the activity and functionality of a urokinase prepared inaccordance to the methods set forth herein are described throughout theliterature, and are similar to assays for other plasminogen and fibrinrelated assays described elsewhere throughout. One example of an assayto determine the activity of an urokinase molecule synthesized asdescribed herein can be as described in, for example, Ploug, et al.(1957, Biochim. Biophys. Acta 24: 278-282), using fibrin platescomprising 1.25% agarose, 4.1 mg/ml human fibrinogen, 0.3 units/ml ofthrombin and 0.5 μg/ml of soybean trypsin inhibitor.

[1224] O. Human DNase

[1225] The present invention further encompasses a method for theremodeling and/or modification of recombinant human DNase. Human DNase Ihas been tested as a therapeutic agent and was shown to diminish theviscosity of cystic fibrosis mucus in vitro. It has been determined thatpurulent mucus contains about 10-13 mg/ml of DNA, an ionic polymerpredicted to affect the rheologic properties of airway fluids.Accordingly, bovine pancreatic DNase I, an enzyme that degrades DNA, wastested as a mucolytic agent many years ago but did not enter clinicalpractice, because of side effects induced by antigenicity and/orcontaminating proteases. Recombinant human DNase is currently used as atherapeutic agent to alleviate the symptoms of diseases such as cysticfibrosis.

[1226] A remodeled rDNase peptide may be administered to a patienthaving cystic fibrosis. A remodeled rDNase peptide may also beadministered to a cystic fibrosis patient to improve pulmonary function.Preferably, the patient is a human patient.

[1227] Similar to DNase derived from bovine sources, recombinant humanDNase poses some problems, mostly due to lowered efficacy due toimproper glycosylation imparted by mammalian expression systemscurrently in use. The present invention describes a method forremodeling DNase, leading to increased efficacy and better therapeuticresults.

[1228] The nucleotide and amino acid sequences of human DNAse arepresented herein as SEQ ID NO:39 and SEQ ID NO:40 (FIGS. 74A and 74B,respectively). Variants of the peptide comprising DNase are well knownin the art. As an example, U.S. Pat. No. 6,348,343 describes a humanDNase with multiple amino acid substitutions throughout the primarystructure. Additionally, U.S. Pat. No. 6,391,607 describes a hyperactivevariant of DNase with multiple amino acid substitutions at positions 9,14, 74, 75, and 205. The present examples, and others well known in theart or to be discovered in the future are encompassed in the presentinvention.

[1229] Expression systems for producing a DNase peptide are well knownto the skilled artisan, and have been described in prokaryotic andeukaryotic systems. For example, PCT Patent Publication No. WO 90/07572describes these methods in considerable detail.

[1230] Assays to determine the biological activity of a DNase moleculedeveloped according to the methods of the present invention are wellknown in the art. As an example, but in no way meant to be limiting tothe present invention, an assay to determine the DNA-hydrolytic activityof human DNase I is presented herein. Briefly, two different plasmiddigestion assays are used. The first assay (“supercoiled DNA digestionassay”) measures the conversion of supercoiled double-stranded plasmidDNA to relaxed (nicked), linear, and degraded forms. The second assay(“linear DNA digestion assay”) measured the conversion of lineardouble-stranded plasmid DNA to degraded forms. Specifically, DNaseprepared according to the methods of the present invention is added to160 microliters of a solution comprising 25 micrograms per milliliter ofeither supercoiled plasmid DNA or EcoRI-digested linearized plasmid DNAin 25 mM HEPES, pH 7.1, 100 μg/ml bovine serum albumin, 1 mM MgCl₂, 2.5mM CaCl₂, 150 mM NaCl, and the samples are incubated at roomtemperature. At various times, aliquots of the reaction mixtures areremoved and quenched by the addition of 25 mM EDTA, together with xylenecyanol, bromophenol blue, and glycerol. The integrity of the plasmid DNAin the quenched samples is analyzed by electrophoresis of the samples onagarose gels. After electrophoresis, the gels are stained with asolution of ethidium bromide and the DNA in the gels is visualized byultraviolet light. The relative amounts of supercoiled, relaxed, andlinear forms of plasmid DNA are determined by scanning the gels with afluorescent imager (such as the Molecular Dynamics Model 575FluorImager) and quantitating the amount of DNA in the bands of the gelthat correspond to the different forms.

[1231] P. Insulin

[1232] The invention further includes a method for remodeling insulin.Insulin is well known as the most effective treatment for type Idiabetes, in which the beta islet cells of the pancreas do not produceinsulin for the regulation of blood glucose levels. The ramifications ofdiabetes and uncontrolled blood glucose include circulatory and footproblems, and blindness, not to mention a variety of other complicationsthat either result from or are exacerbated by diabetes.

[1233] Prior to the cloning and sequencing of human insulin, porcineinsulin was used as a treatment for diabetes. Insulin is now producedrecombinantly, but the short, 51 amino acid sequence of the maturemolecule is a complex structure comprising multiple sulfide bonds.Current methods to recombinantly produce insulin result in a productthat lacks similarity to the native protein as produced in healthynon-diabetic subjects. The present invention seeks to repair this flaw.

[1234] A remodeled insulin peptide may be administered to a patientselected from the group consisting of a patient having Type I Diabetes(diabetes mellitus) and a patient having Type 2 diabetes mellitus whorequires basal (long-acting) insulin for the control of hyperglycemia. Aremodeled insulin peptide may also be administered to a diabetic patientto control hyperglycemia. Preferably, the patient is a human patient.

[1235] The nucleotide and amino acid sequence of human insulin isportrayed in SEQ ID NO:43 and SEQ ID NO:44, respectively (FIGS. 75A and75B, respectively). Variants of insulin are abundant throughout the art.U.S. Pat. No. 6,337,194 describes insulin fusion protein analogs, U.S.Pat. No. 6,323,311 describes insulin derivatives comprising a cyclicanhydride of a dicarboxylic acid, and U.S. Pat. No. 6,251,856 describesan insulin derivative comprising multiple amino acid substitutions and alipophilic group. The skilled artisan will recognize that the followingexamples of insulin derivatives are in no way exhaustive, but simplyrepresent a small sample of those well known in the art. Therefore, thepresent invention comprises insulin derivatives known or to bediscovered.

[1236] Expression systems for the production of insulin are well knownin the art, and can be accomplished using molecular biology techniquesas described in, for example, Sambrook et al. (1989, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York).

[1237] Assays to determine the functionality of an insulin moleculeprepared according to the methods of the present invention are similarlywell known in the art. For example, an in vivo model of glucosedepression can be used to evaluate the biological activity of insulinsynthesized using the methods of the present invention. Useful for thispurpose is a rat model. The animals are fasted overnight (16 hours)prior to the experiment, and then anesthetized with intraperitoneallyadministered sodium pentobarbital or another suitable anesthetic such asketamine. Each animal receives an i.v. injection (tail vein) of theparticular insulin derivative (20 μg/ml/kg). Blood samples are takenfrom the jugular vein 15 and 5 minutes before injection and 15, 30, 60,90, 120, 180, and 240 minutes after injection. Blood glucose levels aremeasured with a blood glucose monitor, available from a variety ofcommercial suppliers.

[1238] Q. Hepatitis B Vaccines (HBsAg)

[1239] The present invention further comprises a method for theremodeling the antigen used in hepatitis B vaccines (HbsAg or HepatitisB sAg). HBsAg is a recombinantly produced surface antigen of thehepatitis B S-protein, and is used to illicit an immune response to thehepatitis B virus, an increasing dangerous virus that results in, amongother things, liver disease including cirrhosis and carcinoma, andresults in over 1 million deaths worldwide annually. Currently the HBsAgvaccine is administered three times over a six month interval to illicita protective and neutralizing immune response.

[1240] HBsAg is currently produced in yeast strains, and thereforereflects the glycosylation patterns native to a fungus. The presentinvention provides a method to remodel HBsAg, resulting in among otherthings, improved immunogenicity, antibodies with improved affinity forthe virus, and the like.

[1241] A remodeled HBsAg peptide may be administered to a patient toimmunize the patient against disease caused by a Hepatitis B virus. Aremodeled HBsAg peptide may also be administered to a predialysispatient or a dialysis patient to immunize the patient against diseasecaused by a Hepatitis B virus. Preferably, the patient is a humanpatient.

[1242] The sequences of the S-protein from a Hepatitis B virus (HBsAg)nucleic acid and primary amino acid chain are set forth herein as SEQ IDNO:45 and SEQ ID NO:46 (FIGS. 76A and 76B, respectively). The nucleotideis 1203 bases in length. The amino acid is 400 residues long. The last226 amino acid residues are the small S-antigen, which is used in theGlaxoSmithKline vaccine and the Merck vaccine. Fifty-five amino acidsupstream from the small S-antigen is the Pre-S start codon. The Pre-S+Sregions are the middle S antigen, which is used in the Aventis Pasteurvaccine. From the first start codon to the Pre-S start codon comprisesthe rest of the S-protein, and is called the large S-protein. This isbut one example of the HBsAg used in vaccines, and other subtypes arewell known, as exemplified in GenBank Acc Nos.: AF415222, AF415221,AF415220, and AF415219. The sequences presented herein are simplyexamples of HBsAg known in the art. Similar antigens have been isolatedfrom other strains of hepatitis B virus, and may or may not have beenevaluated for antigenicity and potential as vaccine candidates. Thepresent invention therefore encompasses hepatitis B vaccine S-proteinsurface antigens known or to be discovered.

[1243] Expression of an HBsAg in an expression system is a routineprocedure for one of skill in the art, and is described in, for example,U.S. Pat. No. 5,851,823. Assays for the immunogenicity of a vaccine arewell known in the art, and comprise various tests for the production ofneutralizing antibodies, and employ techniques such as ELISA,neutralization assays, Western blots, immunoprecipitation, and the like.Briefly, a sandwich ELISA for the detection of effective anti-HBsAgantibodies is described. The Enzygnost HBsAg assay (Aventis Behring,King of Prussia, Pa.) is used for such methods. Wells are coated withanti-HBs. Serum plasma or purified protein and appropriate controls areadded to the wells and incubated. After washing, peroxidase-labeledantibodies to HBsAg are reacted with the remaining antigenicdeterminants. The unbound enzyme-linked antibodies are removed bywashing and the enzyme activity on the solid phase is determined bymethods well known in the art. The enzymatically catalyzed reaction ofhydrogen peroxide and chromogen is stopped by adding diluted sulfuricacid. The color intensity is proportional to the HBsAg concentration ofthe sample and is obtained by photometric comparison of the colorintensity of the unknown samples with the color intensities of theaccompanying negative and positive control sera.

[1244] R. Human Growth Hormone

[1245] The present invention further encompasses a method for theremodeling of human growth hormone (HGH). The isoform of HGH which issecreted in the human pituitary, consists of 191 amino acids and has amolecular weight of about 21,500. The isoform of HGH which is made inthe placenta is a glycosylated form. HGH participates in much of theregulation of normal human growth and development, including lineargrowth (somatogenesis), lactation, activation of macrophages, andinsulin-like and diabetogenic effects, among others.

[1246] HGH is a complex hormone, and its effects are varied as a resultof interactions with various cellular receptors. While compositionscomprising HGH have been used in the clinical setting, especially totreat dwarfism, the efficacy is limited by the absence of glycosylationof the HGH produced recombinantly.

[1247] A remodeled HGH peptide may be administered to a patient selectedfrom the group consisting of a patient having a growth hormonedeficiency, a patient having Turner syndrome, a patient having growthfailure due to a lack of adequate endogenouse growth hormone secretion,a patient having growth failure due to Prader-Willi syndrome (PWS), apatient having growth failure associated with chronic renalinsufficiency, and a patient having AIDS associated wasting or cachexia.A remodeled HGH peptide may also be administered to a patient havingshort stature. Preferably, the patient is a human patient.

[1248] The nucleic and amino acid sequence of HGH are set forthelsewhere herein as SEQ ID NO:47 and SEQ ID NO:48 (FIGS. 77A and 77B,respectively). The skilled artisan will recognize that variants,derivatives, and mutants of HGH are well known. Examples can be found inU.S. Pat. No. 6,143,523 where amino acid residues at positions 10, 14,18, 21, 167, 171, 174, 176 and 179 are substituted, and in U.S. Pat. No.5,962,411 describes splice variants of HGH. The present inventionencompasses these HGH variants known in the art of to be discovered.

[1249] Methods for the expression of HGH in recombinant cells isdescribed in, for example, U.S. Pat. No. 5,795,745. Methods forexpression of HGH in, inter alia, prokaryotes, eukaryotes, insect cellsystems, plants, and in vitro translation systems are well known in theart.

[1250] An HGH molecule produced using the methods of the currentinvention can be assayed for activity using a variety of methods knownto the skilled artisan. For example, U.S. Pat. No. 5,734,024 describes amethod to determine the biological functionality of an expressed HGH.

[1251] S. Anti-Thrombin III

[1252] Antithrombin (antithrombin III, AT-III) is a potent inhibitor ofthe coagulation cascade in blood. It is a non-vitamin K-dependentprotease that inhibits the action of thrombin as well as otherprocoagulant factors (e.g., Factor Xa). Congenital antithrombin IIIdeficiency is an autosomal dominant disorder in which an individualinherits one copy of a defective gene. This condition leads to increasedrisk of venous and arterial thrombosis, with onset of clinicalmanifestations typically presenting in young adulthood. Severecongenital antithrombin III deficiency, in which the individual inheritstwo defective genes, is an autosomal recessive condition associated withincreased thrombogenesis, typically noted in infancy. Acquiredantithrombin III deficiency most commonly is seen in situations wherethere is inappropriate activation of the coagulation system. Commonconditions that result in acquired antithrombin III deficiency includedisseminated intravascular coagulation, microangiopathic hemolyticanemias due to endothelial damage (i.e., Hemolytic-uremic syndrome), andveno-occlusive disease (VOD) seen in patients undergoing bone marrowtransplant. AT-III deficiency may be corrected acutely by infusions ofAT-III concentrates.

[1253] A remodeled AT-III peptide may be administered to a patientselected from the group consisting of a patient having a hereditaryAT-III deficiency in connection with a surgical or obstetrical procedureand a hereditary AT-III deficient patient having a thromboembolism.Preferably, the patient is a human patient.

[1254] Antithrombin III (AT-III) is an α2-glycoprotein of molecularweight 58,000. It is sold commercially as Thrombate III™ (Bayer Corp.,West Haven, Conn.). The nucleic acid and amino acid sequences of humanantithrombin III are displayed in FIGS. 78A (SEQ ID NO:63) and 78B (SEQID NO:64), respectively.

[1255] Methods to make anti-thrombin III are well know to those in theart. For example, published nucleic acid and amino acid sequences areavailable for human antithrombin III (see, U.S. Pat. No. 4,517,294) andmutants of human antithrombin II (see, U.S. Pat. Nos. 5,420,252,5,618,713, 5,700,663). The methods of the invention may be used with anyof these amino acid sequences and any nucleic acid sequences that encodethem, but are not limited to these sequences. Exemplary methods toproduce recombinant antithrombin III are well known in the art, andseveral are described in U.S. Pat. Nos. 5,420,252, 5,843,705, 6,441,145and 5,994,628. Exemplary methods to purify recombinant antithrombin IIIare described in U.S. Pat. Nos. 5,989,593, 6,268,487, 6,395,888,6,395,881, 6,451,978 and 6,518,406.

[1256] There are many known uses for recombinant antithrombin III.Antithrombin III can be used as a anticoagulant during surgery (U.S.Pat. Nos. 5,252,557, 5,182,259), as part of a pharmaceutical preparationor method to inhibit thrombosis (U.S. Pat. Nos. 5,565,471, 6,001,820),and to reduce the adverse side effects of cellular transplantation (U.S.Pat. No. 6,387,366). Additionally, antithrombin III preparations can beused to increase placental blood flow (U.S. Pat. No. 5,888,964), inhibitfertilization (U.S. Pat. No. 5,545,615), treat asthma (U.S. Pat. No.6,355,626) and treat arthritis (U.S. Pat. No. 5,252,557) and otherinflammatory processes (U.S. Pat. No. 6,399,572). Antithrombin III canalso be used to manufacture replacement blood plasma (U.S. Pat. No.4,900,720) or prepare a stabilized cellular blood product (U.S. Pat. No.6,139,878) for transfusions. Antithrombin III may be administered as apharmaceutical preparation (U.S. Pat. Nos. 5,084,273, 5,866,122,6,399,572, 6,156,731 and 6,514,940) or using gene therapy methodology(U.S. Pat. No. 6,410,015). Compositions comprising antithrombin III canbe used as tissue adhesives (U.S. Pat. No. 6,500,427) or lubricants formedical devices that are introduced to the patient (U.S. Pat. No.6,391,832). Antithrombin III can also be used to coat endovascularstents (U.S. Pat. Nos. 6,355,055, 6,240,616, 5,985,307, 5,685,847 and5,222,971), ocular implants (U.S. Pat. No. 5,944,753) and prostheses ingeneral (U.S. Pat. Nos. 6,503,556, 6,491,965 and 6,451,373).Antithrombin III can also be used in methods to locate an internalbleeding site in a patient (U.S. Pat. No. 6,314,314) and to determinehemostatic dysfunction in a patient (U.S. Pat. No. 6,429,017).

[1257] T. Human Chorionic Gonadotropin

[1258] Human Chorionic Gonadotropin (hCG) is a glycoprotein composed ofan alpha subunit and a beta subunit. HCG is closely related to two othergonadotropins, luteinizing hormone (LH) and follicle stimulating hormone(FSH), as well as thyroid stimulating hormone (TSH), all three of whichare glycoprotein hormones. The alpha subunits of these variousglycoprotein hormones are structurally very similar, but the betasubunits differ in amino acid sequence.

[1259] The nucleic acid and amino acid sequences of the human chorionicgonadotropin α-subunit are displayed in FIGS. 79A (SEQ ID NO:69) and 79B(SEQ ID NO:70), respectively. The nucleic acid and amino acid sequencesof the human chorionic gonadotropin β-subunit are displayed in FIGS. 79C(SEQ ID NO:71) and 79D (SEQ ID NO:72), respectively.

[1260] Human chorionic gonadotropin is used in an infertility treatmentto promote ovulation or release of an egg from the ovary in women who donot ovulate on their own. Human chorionic gonadotropin is also given toyoung males to treat undescended or underdeveloped testicles. It is usedin men to stimulate the production of testosterone. Some physicians alsoprescribe human chorionic gonadotropin for men having erictiledysfunctionor lack of sexual desire, and for treatment of male“menopause.”

[1261] A remodeled hCG peptide may be administered to a patient selectedfrom the group consisting of a patient undergoing assisted reproductivetechnology (ART), a patient undergoing in vitro fertilization (IVF), apatient undergoing embryo transfer, an infertile patient, a male patienthaving prepubertal cryptoorchidism not due to anatomical obstruction,and a male patient having hypogonadotropic hypogonadism. A remodeled hCGpeptide may also be administered to induce final follicular maturationand early luteinization in an infertile female patient, wherein theinfertile female patient has undergone pituitary desensitization andpretreatment with follicle stimulating hormones. A remodeled hCG peptidemay also be administered to induce ovulation and pregnancy in ananovulatory infertile patient. Preferably, the patient is a humanpatient.

[1262] Methods to make human chorionic gonadotropin are well known inthe art. The heterodimeric hCG can be recombinantly made in any one ofmany expression systems currently used for industrial manufacture ofrecombinant proteins. One method of making recombinant hCG is describedin U.S. Pat. No. 5,639,639. Methods for making recombinant heterodimericproteins by expressing both subunits in the same cell are, in general,well known in the art, and several methods are described in the U.S.Pat. Nos. 5,643,745 (expression in a filamentous fungus), 5,985,611 and6,087,129 (expression in secretory cells). Alternatively, each subunitcan be expressed individually in cells, and the two subunits laterbrought together in vitro for assembly into the heterdimer.

[1263] Methods for using human chorionic gonadotropin are numerous andwell known in the art. Commonly, hCG is used to induce or synchronizeovulation in mammals (see, U.S. Pat. Nos. 6,489,288, 5,589,457,5,532,155, 4,196,123, 4,062,942 and 4,845,077). Additionally, hCG can beused in pregnancy tests, and in particular agglutination-based tests(see, U.S. Pat. Nos. 3,991,175, 4,003,988, 4,071,314 and 4,088,749). hCGcan also be used in a contraceptive vaccine (see, U.S. Pat. Nos.4,161,519 and 4,966,888). In addition, hCG can be used to treatconditions related to aging and altered hormonal balance such as benignprostatic hypertrophy (see, U.S. Pat. No. 5,610,136) and central nervoussystem diseases common in the elderly (see, U.S. Pat. No. 4,791,099).

[1264] Alternatively, hCG can be used to detect and treat cancers thatexpress hCG or one of its subunits. hCG-expressing tumors include, butare not limited to, breast, prostate, ovary and stomach carcinomas, andneuroblastomas such as Karposi's sarcoma. Antibodies can be raised tohCG which has been glycoremodeled so as to have glycan structuressimilar to those found on the tumor-expressed hCG, and these antibodiesmay be used to detect hCG-expressing tumors in patients according tomethods well known in the art (see, U.S. Pat. Nos. 4,311,688, 4,478,815and 4,323,546). Additionally, remodeled hCG can be used to raise animmune response to a tumor that is expressing hCG (see, U.S. Pat. Nos.5,677,275, 5,762,931, 5,877,148, 4,970,071 and 4,966,753).

[1265] hCG can also be used in methods to generally immunomodulate ananimal, such as described in U.S. Pat. Nos. 5,554,595, 5,851,997 and5,700,781. In addition, hCG can be used as an inhibitor of the matrixmetalloprotease in conditions benefiting from such treatment, such aschronic inflammatory diseases, multiple sclerosis andangiogenesis-dependent diseases (see, U.S. Pat. No. 6,444,639).

[1266] U. α-Iduronidase

[1267] α-Iduronidase is sold commercially as Aldurazyme™ (BioMarin andGenzyme). It is useful for replacement therapy for the treatment of MPSI, a lysosomal storage disease. MPS I (also known as Hurler disease) isa genetic disease caused by the deficiency of alpha-L-iduronidase, anenzyme normally required for the breakdown of certain complexcarbohydrates known as glycosaminoglycans (GAGs). The normal breakdownof GAGs is incomplete or blocked if the enzyme is not present insufficient quantity. The cell is then unable to excrete the carbohydrateresidues and they accumulate in the lysosomes of the cell and cause MPSI.

[1268] A remodeled alpha-iduronidase peptide may be administered to apatient selected from the group consisting of a patient having alysosomal storage disease, a patient having an alpha-L-iduronidasedeficiency, a patient having mucopolysaccaridosis I (MPS I), and apatient having Hurler disease. Preferably, the patient is a humanpatient.

[1269] Methods to produce and purify α-iduronidase, as well as methodsto treat certain genetic disorders including α-L-iduronidase deficiencyand mucopolysaccharidosis I (MPS 1) are described in U.S. Pat. No.6,426,208. The nucleic acid and amino acid sequences of humanα-iduronidase are found in FIGS. 80A (SEQ ID NO:65) and 80B (SEQ IDNO:66), respectively.

[1270] V. α-Galactosidase A

[1271] α-Galactosidase A (also known as agalsidase beta) is soldcommercially as Fabrazyme™ (Genzyme). α-Galactosidase A is useful forthe treatment of Fabry disease. Fabry disease is a rare, inheriteddisorder caused by the deficiency of the essential enzymeα-galactosidase. Without this enzyme, Fabry patients are unable tobreakdown a fatty acid substance in their body calledglobotriasylceramide (GL-3), which accumulates in cells in the bloodvessels of the heart, kidney, brain and other vital organs. Theprogressive buildup of this substance puts patients a risk for stroke,heart attack, kidney damage and debilitating pain. Most patients developkidney failure during adulthood, and severe organ complications lead todeath around age forty.

[1272] A remodeled alpha-galactosidase A peptide may be administered toa patient selected from the group consisting of a patient having alysosomal storage disease, a patient having an alpha-galactosidase Adeficiency, and a patient having Fabry disease. Preferably, the patientis a human patient.

[1273] The α-galactosidase A enzyme is a lysosomal enzyme whichhydrolyzes globotriaosylceramide and related glycolipids which haveterminal α-galactosidase linkages. It is a 45 kDa N-glycosylated proteinencoded on the long arm of the X chromosome. The initial glycosylatedforms (Mr=55,000 to 58,000) synthesized in human fibroblasts or Changliver cells are processed to a mature glycosylated form (Mr=50,000). Themature active enzyme as purified from human tissues and plasma is ahomodimer (Bishop et al., 1986, Proc. Natl. Acad. Sci. USA 83:4859-4863). The nucleic acid and amino acid sequences of α-galactosidaseA are found in FIGS. 81A (SEQ ID NO:67) and 81B (SEQ ID NO:68). Otheruseful nucleic acid and amino acid sequences of alpha-galactosidase Aare found in U.S. Pat. No. 6,329,191.

[1274] References teaching how to make alpha-galactosidase A are foundin U.S. Pat. Nos. 5,179,023 and 5,658,567 (expression in insect cells),U.S. Pat. No. 5,356,804 (expression and secretion from mammalian cells,including CHO cells), U.S. Pat. No. 5,401,451 (expression in mammaliancells), U.S. Pat. No. 5,580,757 (expression in mammalian cells as afusion protein) and U.S. Pat. No. 5,929,304 (expression in plant cells).Methods for purifying recombinant alpha-galactosidase A are found inU.S. Pat. No. 6,395,884.

[1275] References teaching how to use alpha-galactosidase A to treatpatients include, but are not limited to, U.S. Pat. No. 6,066,626 (genetherapy) and U.S. Pat. No. 6,461,609 (treatment with the protein).Mutant forms of alpha-galactosidase A that are useful in the methods ofthe invention include, but are not limited to, those described in U.S.Pat. No. 6,210,666.

[1276] W. Antibodies

[1277] The present invention further comprises a method for theremodeling of various antibody preparations including chimeric antibodypreparations, including, chimeric TNFR, chimeric anti-glycoproteinIIb/IIIa, chimeric anti-HER2, chimeric anti-RSV, chimeric anti-CD20, andchimeric anti-TNF. Chimeric antibody preparations comprise a human Fcportion from an IgG antibody and the variable regions from a monoclonalantibody specific for an antigen. Other preparations comprise areceptor, for example the 75 kDa TNF receptor, fused to a human IgG Feportion. These molecules further include Fab fragments comprising lightand heavy chains from human and mice. A chimeric TNFR is useful in thetreatment of inflammatory diseases, such as rheumatoid arthritis.Chimeric anti-glycoprotein IIb/IIIa is useful in the treatment ofcardiac abnormalities, blood clotting, and platelet functiondisturbances. A chimeric anti-HER2 is useful as a treatment for breastcancer, chimeric anti-RSV is useful for the treatment of respiratorysyncytial virus, chimeric anti-CD20 is useful for the treatment ofNon-Hodgkin's lymphoma, and chimeric anti-TNF is used for treatment ofCrohn's disease.

[1278] While these chimeric antibodies have proved useful in themanagement of varied diseases, administration has to be fairly frequentand at fairly high doses due to the relatively short half-life of arecombinant protein produced in rodent cells. While a majority of thechimeric antibody is human, and therefore regarded as “self” by theimmune system, they are degraded and destroyed due to non-nativeglycosylation patterns. The present invention proposes to repair thisproblem, greatly increasing the efficacy of these novel medicines.

[1279] Antibodies and Methods of Their Generation

[1280] The term “antibody,” as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. Antibodies are typically tetramers ofimmunoglobulin molecules. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, Fv, Fab and F(ab)₂, as well as singlechain antibodies and humanized antibodies (Harlow et al., 1999, UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold SpringHarbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; Bird et al., 1988, Science 242:423-426).

[1281] By the term “synthetic antibody” as used herein, is meant anantibody which is generated using recombinant DNA technology, such as,for example, an antibody expressed by a bacteriophage as describedherein. The term should also be construed to mean an antibody which hasbeen generated by the synthesis of a DNA molecule encoding the antibodyand which DNA molecule expresses an antibody peptide, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

[1282] Monoclonal antibodies directed against full length or peptidefragments of a peptide or peptide may be prepared using any well knownmonoclonal antibody preparation procedures, such as those described, forexample, in Harlow et al. (1988, In: Antibodies, A Laboratory Manual,Cold Spring Harbor, N.Y.) and in Tuszynski et al. (1988, Blood,72:109-115). Quantities of the desired peptide may also be synthesizedusing chemical synthesis technology. Alternatively, DNA encoding thedesired peptide may be cloned and expressed from an appropriate promotersequence in cells suitable for the generation of large quantities ofpeptide. Monoclonal antibodies directed against the peptide aregenerated from mice immunized with the peptide using standard proceduresas referenced herein.

[1283] Nucleic acid encoding the monoclonal antibody obtained using theprocedures described herein may be cloned and sequenced using technologywhich is available in the art, and is described, for example, in Wrightet al. (1992, Critical Rev. in Immunol. 12(3,4):125-168) and thereferences cited therein. Further, the antibody of the invention may be“humanized” using the technology described in Wright et al., (supra) andin the references cited therein, and in Gu et al. (1997, Thrombosis andHematocyst 77(4):755-759).

[1284] To generate a phage antibody library, a cDNA library is firstobtained from mRNA which is isolated from cells, e.g., the hybridoma,which express the desired peptide to be expressed on the phage surface,e.g., the desired antibody. cDNA copies of the mRNA are produced usingreverse transcriptase. cDNA which specifies immunoglobulin fragments areobtained by PCR and the resulting DNA is cloned into a suitablebacteriophage vector to generate a bacteriophage DNA library comprisingDNA specifying immunoglobulin genes. The procedures for making abacteriophage library comprising heterologous DNA are well known in theart and are described, for example, in Sambrook and Russell (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.).

[1285] Bacteriophage which encode the desired antibody, may beengineered such that the peptide is displayed on the surface thereof insuch a manner that it is available for binding to its correspondingbinding peptide, e.g., the antigen against which the antibody isdirected. Thus, when bacteriophage which express a specific antibody areincubated in the presence of a cell which expresses the correspondingantigen, the bacteriophage will bind to the cell. Bacteriophage which donot express the antibody will not bind to the cell. Such panningtechniques are well known in the art and are described for example, inWright et al., (supra).

[1286] Processes such as those described above, have been developed forthe production of human antibodies using M13 bacteriophage display(Burton et al., 1994, Adv. Immunol. 57:191-280). Essentially, a cDNAlibrary is generated from mRNA obtained from a population ofantibody-producing cells. The mRNA encodes rearranged immunoglobulingenes and thus, the cDNA encodes the same. Amplified cDNA is cloned intoM13 expression vectors creating a library of phage which express humanantibody fragments on their surface. Phage which display the antibody ofinterest are selected by antigen binding and are propagated in bacteriato produce soluble human immunoglobulin. Thus, in contrast toconventional monoclonal antibody synthesis, this procedure immortalizesDNA encoding human immunoglobulin rather than cells which express humanimmunoglobulin.

[1287] Remodeling Glycans of Antibody Molecules

[1288] The specific glycosylation of one class of peptides, namelyimmunoglobulins, has a particularly important effect on the biologicalactivity of these peptides. The invention should not be construed to belimited solely to immunoglobulins of the IgG class, but should also beconstrued to include immunoglobulins of the IgA, IgE and IgM classes ofantibodies.

[1289] Further, the invention should not be construed to be limitedsolely to any type of traditional antibody structure. Rather, theinvention should be construed to include all types of antibodymolecules, including, for example, fragments of antibodies, chimericantibodies, human antibodies, humanized antibodies, etc.

[1290] A typical immunoglobulin molecule comprises an effector portionand an antigen binding portion. For a review of immunoglobulins, seeHarlow et al., 1988, Antibodies: A Laboratory Manual, Cold SpringHarbor, N.Y., and Harlow et al., 1999, Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY. The effector portion ofthe immunoglobulin molecule resides in the Fc portion of the moleculeand is responsible in part for efficient binding of the immunoglobulinto its cognate cellular receptor. Improper glycosylation ofimmunoglobulin molecules particularly in the CH2 domain of the Fcportion of the molecule, affects the biological activity of theimmunoglobulin.

[1291] More specifically with respect to the immunoglobulin IgG, IgGeffector function is governed in large part by whether or not the IgGcontains an N-acetylglucosamine (GlcNAc) residue attached at the 4-Oposition of the branched mannose of the trimannosyl core of the N-glycanat Asparagine (Asn) 297 in the CH2 domain of the IgG molecule. Thisresidue is known as a “bisecting GlcNAc.” The purpose of addingbisecting GlcNAc to the N-glycan chains of a natural or recombinant IgGmolecule or a IgG-Fc-containing chimeric construct is to optimize Fcimmune effector function of the Fc portion of the molecule. Sucheffector functions may include antibody-dependent cellular cytotoxicity(ADCC) and any other biological effects that require efficient bindingto FcγR receptors, and binding to the C1 component of complement. Theimportance of bisecting GlcNAc for achieving maximum immune effectorfunction of IgG molecules has been described (Lifely et al., 1995,Glycobiology 5 (8): 813-822; Jeffris et al., 1990, Biochem. J. 268 (3):529-537).

[1292] The glycans found at the N-glycosylation site at Asn 297 in theCH2 domain of IgG molecules have been structurally characterized for IgGmolecules found circulating in human and animal blood plasma, IgGproduced by myeloma cells, hybridoma cells, and a variety of transfectedimmortalized mammalian and insect cell lines. In all cases the N-glycanis either a high mannose chain or a complete (Man3, GlcNAc4, Gal2,NeuAc2, Fuc1) or variably incomplete biantennary chain with or withoutbisecting GlcNAc (Raju et al., 2000, Glycobiology 10 (5): 477-486;Jeffris et al., 1998, Immunological. Rev. 163L59-76; Lerouge et al.,1998, Plant Mol. Biol. 38: 31-48; James et al., 1995, Biotechnology 13:592-596).

[1293] The present invention provides an in vitro customizedglycosylated immunoglobulin molecule. The immunoglobulin molecule may beany immunoglobulin molecule, including, but not limited to, a monoclonalantibody, a synthetic antibody, a chimeric antibody, a humanizedantibody, and the like. Specific methods of generating antibodymolecules and their characterization are disclosed elsewhere herein.Preferably, the immunoglobulin is IgG, and more preferably, the IgG is ahumanized or human IgG, most preferably, IgG1.

[1294] The present invention specifically contemplates usingβ1,4-mannosyl-glycopeptide β1,4-N-acetylglucosaminyltransferase,GnT-III: EC2.4.1.144 as an in vitro reagent to glycosidically linkN-acetylglucosamine (GlcNAc) onto the 4-O position of the branchedmannose of the trimannosyl core of the N-glycan at Asn 297 in the CH2domain of an IgG molecule. However, as will be appreciated from thedisclosure provided herein, the invention should not be construed tosolely include the use of this enzyme to provide a bisecting GlcNAc toan immunoglobulin molecule. Rather, it has been discovered that it ispossible to modulate the glycosylation pattern of an antibody moleculesuch that the antibody molecule has enhanced biological activity, i.e.,effector function, in addition to potential enhancement of otherproperties, e.g., stability, and the like.

[1295] There is provided in the present invention a general method forremoving fucose molecules from the Asn(297) N-linked glycan for thepurpose of enhancing binding to Fc-gammaRIIIA, and enhancedantibody-dependent cellular cytotoxicity (see, Shields et al., 2002, J.Biol. Chem. 277:26733-26740). The method entails contacting the antibodymolecule with a fucosidase appropriate for the linkage of the fucosemolecule(s) on the antibody glycan(s). Alternately, the recombinantantibody can be expressed in cells that do express fucosyltransferases,such as the Lec13 varient of CHO cells. The removal of fucose from theglycan(s) of the antibody can be done alone, or in conjunction withother methods to remodel the glycans, such as adding a bisecting GlcNAc.Expression of antibodies in cells lacking GnT-I may also result in Fcglycans lacking core fucose, which can be further modified by thepresent invention.

[1296] There is provided in the present invention a general method forintroducing a bisecting GlcNAc for the purpose of enhancing Fc immuneeffector function in any preparation of IgG molecules containingN-linked oligosaccharides in the CH2 domain, typically at Asn 297. Themethod requires that the population of IgG molecules is brought to astate of glycosylation such that the glycan chain is an acceptor forGnT-III. This is accomplished in any one of three ways: 1) by selectionor genetic manipulation of a host expression system that secretes IgGwith N-glycan chains that are substrates for GnT-III; 2) by treatment ofa population of IgG glycoforms with exoglycosidases such that the glycanstructure(s) remaining after exoglycosidase treatment is an acceptor forGnT-III; 3) some combination of host selection and exoglycosidasetreatment as in 1) and 2) above plus successive additions of GlcNAc byGnT-I and GnT-III to create an acceptor for GnT-III.

[1297] For example, IgG obtained from chicken plasma contains primarilyhigh mannose chains and would require digestion with one or moreα-mannosidases to create a substrate for addition of GlcNAc to the α1,3mannose branch of the trimannosyl core by GnT-I. This substrate could bethe elemental trimannosyl core, Man3GlcNAc2. Treatment of this corestructure with a combination of GnT-I, GnT-II, and GnT-III usingUDP-GlcNAc as a sugar donor creates Man3GlcNAc5 as shown in FIG. 1. Theorder of action of these glycosyltransferases may be varied to optimizethe production of the desired product. Optionally, this structure canthen be extended by treatment with β1,4 galactosyltransferase. Ifrequired, the galactosylated oligosaccharide can be further extendedusing α2,3- or α2,6-sialyltransferase to achieve a completed biantennarystructure. Using this method biantennary glycan chains can be remodeledas required for the optimal Fc immune effector function of anytherapeutic IgG under development (FIG. 3).

[1298] Alternatively, IgG molecules found in the plasma of most animalsor IgG which is secreted as a recombinant product by most animal cellsor by transgenic animals typically include a spectrum of biantennaryglycoforms including complete (NeuAc2, Gal2, GlcNAc4, Man3, ±Fuc1) (FIG.3) and variably incomplete forms, with or without bisecting GlcNAc (Rajuet al., 2000, Glycobiology 10 (5): 477-486; Jeffris et al., 1998,Immunological Rev. 163: 59-76). To ensure that bisecting GlcNAc ispresent in the entire population of immunoglobulin molecules soproduced, the mixture of molecules can be treated with the followingexoglycosidases, successively or in a mixture: neuraminidase,β-galactosidase, β-hexosaminidase, α-fucosidase. The resultingtrimannosyl core can then be remodeled using glycosyltransferases asnoted above.

[1299] In some cases it may be desired to abolish effector function fromexisting antibody molecules. The present invention also includesmodifying the Fc glycans with appropriate glycosidases andglycosyltransferases to eliminate effector function. Also anticipated isthe addition of sugars modified with PEG or other polymers that serve tohinder or abolish binding of Fc receptors or complement to the antibody.

[1300] In addition, IgG secreted by transgenic animals or stored as“plantibodies” by transgenic plants have been characterized. An IgGmolecule produced in a transgenic plant having N-glycans that containβ1,2 linked xylose and/or α1,3 linked fucose can be treated withexoglycosidases to remove those residues, in addition to the abovedescribed exoglycosidases in order to create the trimannosyl core or aMan3GlcNAc4 structure, and are then treated with glycosyltransferases toremodel the N-glycan as described above.

[1301] The primary novel aspect of the current invention is theapplication of appropriate glycosyltransferases, with or without priorexoglycosidase treatment, applied in the correct sequence to optimizethe effector function of the antibody. In one exemplary embodiment, abisecting GlcNAc is introduced into the glycans of IgG molecules or orother IgG-Fc-chimeric constructs where bisecting GlcNAc is required. Inanother exemplary embodiment, the core fucose is removed from theglycans of IgG molecules or other IgG-Fc-chimeric constructs.

[1302] X. TNF Receptor-IgG Fc Fusion Protein

[1303] The nucleotide and amino acid sequences of the 75 kDa human TNFreceptor are set forth herein as SEQ ID NO:31 and SEQ ID NO:32,respectively (FIGS. 82A and 82B, respectively). The amino acid sequencesof the light and heavy variable regions of chimeric anti-HER2 are setforth as SEQ ID NO:35 and SEQ ID NO:36, respectively (FIGS. 83A and 83B,respectively). The amino acid sequences of the heavy and light variableregions of chimeric anti-RSV are set forth as SEQ ID NO:38 and SEQ IDNO:37, respectively (FIGS. 84A and 84B, respectively). The amino acidsequences of the non-human variable regions of anti-TNF are set forthherein as SEQ ID NO:41 and SEQ ID NO:42, respectively (FIGS. 85A and85B, respectively). The nucleotide and amino acid sequence of the Fcportion of human IgG is set forth as SEQ ID NO:49 and SEQ ID NO:50(FIGS. 86A and 86B, respectively).

[1304] A remodeled chimeric ENBREL™ may be administered to a patientselected from the group consisting of a patient having rheumatoidarthritis and a patient having polyarticular-course juvenile arthritis.A remodeled chimeric ENBREL™ may also be administered to an arthritispatient to reduce signs, symptoms, or structural damage in the patient.Preferably, the patient is a human patient.

[1305] A remodeled Synagis™ antibody may be administered to a patient toimmunize the patient against infection by respiratory syncytial virus(RSV). A remodeled Synagis™ antibody may also be administered to apatient to prevent or reduce the severity of a lower respiratory tractdisease caused by RSV. Preferably, the patient is a human patient.

[1306] Y. MAb Anti-Glycoprotein IIb/IIIa

[1307] The amino acid sequences of a murine anti-glycoprotein IIb/IIIaantibody variable regions are set forth in SEQ ID NO:52 (murine maturevariable light chain, FIG. 87) and SEQ ID NO: 54 (murine mature variableheavy chain, FIG. 88). These murine sequences can be combined with humanIgG amino acid sequences SEQ ID NO:51 (human mature variable lightchain, FIG. 89), SEQ ID NO: 53 (human mature variable heavy chain, FIG.90), SEQ ID NO: 55 (human light chain, FIG. 91) and SEQ ID NO: 56 (humanheavy chain, FIG. 92) according to the proceedures found in U.S. Pat.No. 5,777,085 to create a chimeric humanized murine anti-glycoproteinIIb/IIa antibody. Other anti-glycoprotein IIb/IIIa humanized antibodiesare found in U.S. Pat. No. 5,877,006. A cell line expressing theanti-glycoprotein IIb/IIIa MAb 7E3 can be commercially obtained from theATCC (Manassas, Va.) as accession no. HB-8832.

[1308] Indications for Selected Antibodies

[1309] A remodeled Reopro™ may be administered to a patient selectedfrom the group consisting of a patient undergoing percutaneous coronaryintervention and a patient having unstable angina, wherein the patientis scheduled for percutaneous coronary intervention within 24 hours. Aremodeled Reopro™ may also be administered to a patient undergoingpercutaneous coronary intervention to reduce or prevent a cardiacischemic complication in the patient. Preferably, the patient is a humanpatient.

[1310] A remodeled Herceptin™ may be administered to a patient havingmetastatic breast cancer that overexpresses the HER2 protein.Preferably, the patient is a human patient.

[1311] A remodeled Remicade™ antibody may be administered to a patientselected from the group consisting of a patient having rheumatoidarthritis, a patient having Crohn's disease, and a patient havingfistulizing Crohn's disease. A remodeled Remicade™ antibody may also beadministered to a rheumatoid arthritis patient to reduce signs andsymptoms of rheumatoid arthritis in the patient. A remodeled Remicade™antibody may also be administered to a Crohn's disease patient to reducesigns and symptoms of Crohn's disease in the patient. Preferably, thepatient is a human patient.

[1312] Z. MAb Anti-CD20

[1313] The nucleic acid and amino acid sequences of a chimeric anti-CD20antibody are set forth in SEQ ID NO: 59 (nucleic acid sequence of murinevariable region light chain, FIG. 93A), SEQ ID NO:60 (amino acidsequence of murine variable region light chain, FIG. 93B), SEQ ID NO:61(nucleic acid sequence of murine variable region heavy chain, FIG. 94A)and SEQ ID NO:62 (amino acid sequence of murine variable region heavychain, FIG. 94B). In order to humanize a murine antibody, the TCAE 8(SEQ ID NO:57, FIGS. 95A-95E), which contains the human IgG heavy andlight constant domains, may be conveniently used. By cloning the abovemurine variable region encoding DNA into the TCAE 8 vector according toinstructions given in U.S. Pat. No. 5,736,137, a vector is created (SEQID NO: 58, FIGS. 96A-96E) which when transformed into a mammaliam cellline, expresses a chimeric anti-CD20 antibody. Other humanized anti-CD20antibodies are found in U.S. Pat. No. 6,120,767. A cell line expressingthe anti-CD20 MAb C273 can be commercially obtained from the ATCC(Manassas, Va.) as accession no. HB-9303.

[1314] The skilled artisan will readily appreciate that the sequencesset forth herein are not exhaustive, but are rather examples of thevariable regions, receptors, and other binding moieties of chimericantibodies. Further, methods to construct chimeric or “humanized”antibodies are well known in the art, and are described in, for example,U.S. Pat. No. 6,329,511 and U.S. Pat. No. 6,210,671. Coupled with thepresent disclosure and methods well known throughout the art, theskilled artisan will recognize that the present invention is not limitedto the sequences disclosed herein.

[1315] The expression of a chimeric antibody is well known in the art,and is described in detail in, for example, U.S. Pat. No. 6,329,511.Expression systems can be prokaryotic, eukaryotic, and the like.Further, the expression of chimeric antibodies in insect cells using abaculovirus expression system is described in Putlitz et al. (1990,Bio/Technology 8:651-654). Additionally, methods of expressing a nucleicacid encoding a fusion or chimeric protein are well known in the art,and are described in, for example, Sambrook et al. (2001, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NewYork) and Ausubel et al. (1997, Current Protocols in Molecular Biology,Green & Wiley, New York).

[1316] Determining the function and biological activity of a chimericantibody produced according to the methods of the present invention is asimilarly basic operation for one of skill in the art. Methods fordetermining the affinity of an antibody by competition assays aredetailed in Berzofsky (J. A. Berzofsky and I. J. Berkower, 1984, inFundamental Immunology (ed. W. E. Paul), Raven Press (New York), 595).Briefly, the affinity of the chimeric antibody is compared to that ofthe monoclonal antibody from which it was derived using aradio-iodinated monoclonal antibody.

[1317] A remodeled anti-CD20 antibody may be administered to a patienthaving relapsed or refractory low grade or follicular, CD20-positive,B-cell non-Hodgkin's lymphoma. Preferably, the patient is a humanpatient.

[1318] VII. Pharmaceutical Compositions

[1319] In another aspect, the invention provides a pharmaceuticalcomposition. The pharmaceutical composition includes a pharmaceuticallyacceptable diluent and a covalent conjugate between anon-naturally-occurring, water-soluble polymer, therapeutic moiety orbiomolecule and a glycosylated or non-glycosylated peptide. The polymer,therapeutic moiety or biomolecule is conjugated to the peptide via anintact glycosyl linking group interposed between and covalently linkedto both the peptide and the polymer, therapeutic moiety or biomolecule.

[1320] Pharmaceutical compositions of the invention are suitable for usein a variety of drug delivery systems. Suitable formulations for use inthe present invention are found in Remington's Pharmaceutical Sciences,Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a briefreview of methods for drug delivery, see, Langer, Science 249:1527-1533(1990).

[1321] The pharmaceutical compositions may be formulated for anyappropriate manner of administration, including for example, topical,oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactate polyglycolate) may also be employed ascarriers for the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268 and 5,075,109.

[1322] Commonly, the pharmaceutical compositions are administeredparenterally, e.g., intravenously. Thus, the invention providescompositions for parenteral administration which comprise the compounddissolved or suspended in an acceptable carrier, preferably an aqueouscarrier, e.g., water, buffered water, saline, PBS and the like. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH adjusting and buffering agents, tonicity adjusting agents, wettingagents, detergents and the like.

[1323] These compositions may be sterilized by conventionalsterilization techniques, or may be sterile filtered. The resultingaqueous solutions may be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile aqueous carrierprior to administration. The pH of the preparations typically will bebetween 3 and 11, more preferably from 5 to 9 and most preferably from 7and 8.

[1324] In some embodiments the peptides of the invention can beincorporated into liposomes formed from standard vesicle-forming lipids.A variety of methods are available for preparing liposomes, as describedin, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9: 467 (1980), U.S.Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. The targeting of liposomesusing a variety of targeting agents (e.g., the sialyl galactosides ofthe invention) is well known in the art (see, e.g., U.S. Pat. Nos.4,957,773 and 4,603,044).

[1325] Standard methods for coupling targeting agents to liposomes canbe used. These methods generally involve incorporation into liposomes oflipid components, such as phosphatidylethanolamine, which can beactivated for attachment of targeting agents, or derivatized lipophiliccompounds, such as lipid-derivatized peptides of the invention.

[1326] Targeting mechanisms generally require that the targeting agentsbe positioned on the surface of the liposome in such a manner that thetarget moieties are available for interaction with the target, forexample, a cell surface receptor. The carbohydrates of the invention maybe attached to a lipid molecule before the liposome is formed usingmethods known to those of skill in the art (e.g., alkylation oracylation of a hydroxyl group present on the carbohydrate with a longchain alkyl halide or with a fatty acid, respectively). Alternatively,the liposome may be fashioned in such a way that a connector portion isfirst incorporated into the membrane at the time of forming themembrane. The connector portion must have a lipophilic portion, which isfirmly embedded and anchored in the membrane. It must also have areactive portion, which is chemically available on the aqueous surfaceof the liposome. The reactive portion is selected so that it will bechemically suitable to form a stable chemical bond with the targetingagent or carbohydrate, which is added later. In some cases it ispossible to attach the target agent to the connector molecule directly,but in most instances it is more suitable to use a third molecule to actas a chemical bridge, thus linking the connector molecule which is inthe membrane with the target agent or carbohydrate which is extended,three dimensionally, off of the vesicle surface. The dosage ranges forthe administration of the peptides of the invention are those largeenough to produce the desired effect in which the symptoms of the immuneresponse show some degree of suppression. The dosage should not be solarge as to cause adverse side effects. Generally, the dosage will varywith the age, condition, sex and extent of the disease in the animal andcan be determined by one of skill in the art. The dosage can be adjustedby the individual physician in the event of any counterindications.

[1327] Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achieved bythe use of polymers to conjugate, complex or adsorb the peptide. Thecontrolled delivery may be exercised by selecting appropriatemacromolecules (for example, polyesters, polyaminocarboxymethylcellulose, and protamine sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled release preparations is to incorporate the peptideinto particles of a polymeric material such as polyesters, polyaminoacids, hydrogels, poly (lactic acid) or ethylene vinylacetatecopolymers.

[1328] In order to protect peptides from binding with plasma proteins,it is preferred that the peptides be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly (methymethacrylate) microcapsules,respectively, or in colloidal drug delivery systems, for example,liposomes, albumin microspheres, microemulsions, nanoparticles, andnanocapsules or in macroemulsions. Such teachings are disclosed inRemington's Pharmaceutical Sciences (16th Ed., A. Oslo, ed., Mack,Easton, Pa., 1980).

[1329] The peptides of the invention are well suited for use intargetable drug delivery systems such as synthetic or natural polymersin the form of macromolecular complexes, nanocapsules, microspheres, orbeads, and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, liposomes, and resealed erythrocytes. Thesesystems are known collectively as colloidal drug delivery systems.Typically, such colloidal particles containing the dispersed peptidesare about 50 nm-2 μm in diameter. The size of the colloidal particlesallows them to be administered intravenously such as by injection, or asan aerosol. Materials used in the preparation of colloidal systems aretypically sterilizable via filter sterilization, nontoxic, andbiodegradable, for example albumin, ethylcellulose, casein, gelatin,lecithin, phospholipids, and soybean oil. Polymeric colloidal systemsare prepared by a process similar to the coacervation ofmicroencapsulation.

[1330] In an exemplary embodiment, the peptides are components of aliposome, used as a targeted delivery system. When phospholipids aregently dispersed in aqueous media, they swell, hydrate, andspontaneously form multilamellar concentric bilayer vesicles with layersof aqueous media separating the lipid bilayer. Such systems are usuallyreferred to as multilamellar liposomes or multilamellar vesicles (MLVs)and have diameters ranging from about 100 nm to about 4 μm. When MLVsare sonicated, small unilamellar vesicles (SUVS) with diameters in therange of from about 20 to about 50 nm are formed, which contain anaqueous solution in the core of the SUV.

[1331] Examples of lipids useful in liposome production includephosphatidyl compounds, such as phosphatidylglycerol,phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine.Particularly useful are diacylphosphatidylglycerols, where the lipidmoiety contains from 14-18 carbon atoms, particularly from 16-18 carbonatoms, and are saturated. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine, anddistearoylphosphatidylcholine.

[1332] In preparing liposomes containing the peptides of the invention,such variables as the efficiency of peptide encapsulation, lability ofthe peptide, homogeneity and size of the resulting population ofliposomes, peptide-to-lipid ratio, permeability instability of thepreparation, and pharmaceutical acceptability of the formulation shouldbe considered. Szoka, et al, Annual Review of Biophysics andBioengineering, 9: 467 (1980); Deamer, et al., in LiposoMEs, MarcelDekker, New York, 1983, 27: Hope, et al., Chem. Phys. Lipids, 40: 89(1986)).

[1333] The targeted delivery system containing the peptides of theinvention may be administered in a variety of ways to a host,particularly a mammalian host, such as intravenously, intramuscularly,subcutaneously, intra-peritoneally, intravascularly, topically,intracavitarily, transdermally, intranasally, and by inhalation. Theconcentration of the peptides will vary upon the particular application,the nature of the disease, the frequency of administration, or the like.The targeted delivery system-encapsulated peptide may be provided in aformulation comprising other compounds as appropriate and an aqueousphysiologically acceptable medium, for example, saline, phosphatebuffered saline, or the like.

[1334] The compounds prepared by the methods of the invention may alsofind use as diagnostic reagents. For example, labeled compounds can beused to locate areas of inflammation or tumor metastasis in a patientsuspected of having an inflammation. For this use, the compounds can belabeled with ¹²⁵I, ¹⁴C, or tritium.

EXPERIMENTAL EXAMPLES

[1335] The invention is now described with reference to the followingExamples. These Examples are provided for the purpose of illustrationonly and the invention should in no way be construed as being limited tothese Examples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

[1336] The materials and methods used in the experiments presented inthis Example are now described.

[1337] A. General Procedures

[1338] 1. Preparation of CMP-SA-PEG

[1339] This example sets forth the preparation of CMP-SA-PEG.

[1340] Preparation of2-(benzyloxycarboxamido)-glycylamido-2-deoxy-D-mannopyranose.N-benzyloxycarbonyl-glycyl-N-hydroxysuccinimide ester (3.125 g, 10.2mmol) was added to a solution containing D-mannosamine-HCl (2 g, 9.3mmol) and triethylamine (1.42 mL, 10.2 mmol) dissolved in MeOH (10 mL)and H₂O (6 mL). The reaction was stirred at room temperature for 16hours and concentrated using rotoevaporation. Chromatography (silica,10% MeOH/CH₂Cl₂) yielded 1.71 g (50% yield) of product as a white solid:R_(f)=0.62 (silica; CHCl₃:MeOH:H₂O, 6/4/1); ¹H NMR (CD₃OD, 500 MHz) δ3.24-3.27 (m, 2H), 3.44 (t, 1H), 3.55 (t, 1H), 3.63-3.66 (m, 1H),3.76-3.90 (m, 6H), 3.91 (s, 2H), 4.0 (dd, 2H), 4.28 (d, 1H, J=4.4), 4.41(d, 1H, J=3.2), 5.03 (s, 1H), 5.10 (m, 3H), 7.29-7.38 (m, 10H).

[1341] Preparation of5-(N-benzyloxycarboxamido)glycylamido-3,5-dideoxy-D-glycero-D-galacto-2-nonulopyranosuronate.2-(N-Benzyloxycarboxamido) glycylamide-2-deoxy-D-mannopyranose (1.59 g,4.3 mmol) was dissolved in a solution of 0.1 M HEPES (12 mL, pH 7.5) andsodium pyruvate (4.73 g, 43 mmol). Neuraminic acid aldolase (540 U ofenzyme in 45 mL of a 10 mM phosphate buffered solution containing 0.1 MNaCl at pH 6.9) and the reaction mixture was heated to 37° C. for 24 hr.The reaction mixture was then centrifuged and the supernatant waschromatographed (C18 silica, gradient from H₂O (100%) to 30%MeOH/water). Appropriate fractions were pooled, concentrated and theresidue chromatographed (silica, gradient from 10% MeOH/CH₂Cl₂ toCH₂Cl₂/MeOH/H2O 6/4/1). Appropriate fractions were collected,concentrated and the residue resuspended in water. After freeze-drying,the product (1.67 g, 87% yield) was obtained as a white solid:R_(f)=0.26 (silica, CHCl₃/MeOH/H₂O 6/4/1); ¹H NMR (D20, 500 MHz) δ 1.82(t, 1H), 2.20 (m, 1H), 3.49 (d, 1H), 3.59 (dd, 1H), 3.67-3.86 (m, 2H),3.87 (s, 2H), 8.89-4.05 (m, 3H), 5.16 (s, 2H), 7.45 (m, 5H).

[1342] Preparation of5-glycylamido-3,5-dideoxy-D-glycero-D-galacto-2-nonulopyranosuronate.5-(N-Benzyloxycarboxamido)glycylamido-3,5-dideoxy-D-glycero-D-galacto-2-nonulopyranosuronate(1.66 g, 3.6 mmol) was dissolved in 20 mL of 50% water/methanol. Theflask was repeatedly evacuated and placed under argon and then 10% Pd/C(0.225 g) was added. After repeated evacuation, hydrogen (about 1 atm)was then added to the flask and the reaction mixture stirred for 18 hr.The reaction mixture was filtered through celite, concentrated by rotaryevaporation and freeze-dried to yield 1.24 g (100% yield) of product asa white solid: R_(f)=0.25 (silica, IPA/H₂O/NH₄OH 7/2/1); ¹H NMR (D20,500 MHz) δ 1.83 (t, 1H, J=9.9), 2.23 (dd, 1H, J=12.9, 4.69), 3.51-3.70(m, 2H), 3.61 (s, 2H), 3.75-3.84 (m, 2H), 3.95-4.06 (m, 3H).

[1343] Preparation ofcytidine-5′-monophosphoryl-[5-(N-fluorenylmethoxy-carboxamido)glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate].A solution containing5-glycylamido-3,5-dideoxy-D-glycero-D-galacto-2-nonulopyranosuronate(0.55 g, 1.70 mmol) dissolved in 20 mL H₂O was added to a solution ofTris (1.38 g, 11.4 mmol), 1 M MgCl₂ (1.1 mL) and BSA (55 mg). The pH ofthe solution was adjusted to 8.8 with 1M NaOH (2 mL) and CTP-2Na⁺ (2.23g, 4.2 mmol) was added. The reaction mixture pH was controlled with a pHcontroller which delivered 1 M NaOH as needed to maintain pH 8.8. Thefusion protein (sialyltransferase/CMP-neuraminic acid synthetase) wasadded to the solution and the reaction mixture was stirred at roomtemperature. After 2 days, an additional amount of fusion protein wasadded and the reaction stirred an additional 40 hours. The reactionmixture was precipitated in EtOH and the precipitate was washed 5 timeswith cold EtOH to yield 2.3 grams of a white solid. About 1.0 g of thecrude product was dissolved in 1,4 dioxane (4 mL), H₂O (4 mL) andsaturated NaHCO₃ (3 mL) and a solution of FMOC-C1 (308 mg, 1.2 mmol)dissolved in 2 ml dioxane was added dropwise. After stirring for 16 hrat room temperature, the reaction mixture was concentrated to about 6 mLby rotary evaporation and purified using chromatography (C18 silica,gradient 100% H₂₀ to 30% MeOH/H₂O). Appropriate fractions were combinedand concentrated. The residue was dissolved in water and freeze-dried toyield 253 mg of a white solid: R_(f)=0.50 (silica, IPA/H200NH₄OH 7/2/1);¹H NMR (D20, 500 MHz) δ 1.64 (dt, 1H, J=12.0, 6.0), 2.50 (dd, 1H,J=13.2, 4.9), 3.38 (d, J=9.67, 1H), 3.60 (dd, J=11.65, 6.64, 1H), 3.79(d, J=4.11, 1H), 3.87 (dd, J=12.24, 1.0, 1H), 3.97 (m, 2H), 4.07 (td,J=10.75, 4.84, 1H), 4.17 (dd, J=10.68, 1.0, 1H), 4.25 (s, 2H), 4.32 (t,J=4.4, 1H), 4.37 (t, J=5.8 1H), 4.6-4.7 (m, obscured by solvent peak),5.95 (d, J=4, 1H), 6.03 (d, J=7.4, 1H), 7.43-7.53 (m, 3H), 7.74 (m, 2H),7.94 (q, J=7, 3H). MS (ES); calc. for C₃₅H₄₂N₅O₁₈P ([M−H]⁻), 851.7;found 850.0.

[1344] Preparation ofcytidine-5′-monophosphoryl-(5-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate).Diisopropylamine (83 uL, 0.587 μmol) was added to a solution ofcytidine-5′-monophosphoryl-[5-(N-fluorenyl-methoxycarboxamido)glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate](100 mg, 0.117 mmol) dissolved in water (3 mL) and methanol (1 mL). Thereaction mixture was stirred 16 hr at room temperature and the reactionmethanol removed from the reaction mixture by rotary evaporation. Thecrude reaction mixture was filtered through a C18 silica gel columnusing water and the efluant was collected and freeze-dried to yield (87mg, 100%) of product as a white solid: R_(f)=0.21 (silica, IPA/H₂O/NH₄OH7/2/1); ¹H NMR (D₂O, 500 MHz) δ 1.66 (td, 1H, J=5.3), 2.50 (dd, 1H,J=13.2, 4.6), 3.43 (d, J=9.58, 1H), 3.63 (dd, J=11.9, 6.44, 1H), 3.88(dd, J=11.8, 1.0, 1H), 3.95 (td, J=9.0, 2.3, 1H), 4.10 (t, J=10.42, 1H),4.12 (td, J=10.34, 4.66, 1H), 4.18 (d, J=10.36, 1H), 4.24 (m, 2H), 4.31(t, J=4.64, 1H), 4.35 (t, 1H), 6.00 (d, J=4.37, 1H), 6.13 (d, J=7.71,1H), 7.98 (d, J=7.64, 1H). MS (ES); calc. for C₂₁H₃₂N₅O₁₁P ([M−H]⁻),629.47; found 627.9.

[1345] Preparation ofcytidine-5′-monophosphoryl-[5-(N-methoxy-polyoxyethylene-(1kDa)-3-oxypropionamido)-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate].Benzyltriazol-1-yloxy-tris(dimethylamino)-phosphoniumhexafluorophosphate (BOP, 21 mg, 48 μmol) was added to a solution ofmethoxypolyoxyethylene-(1 kDa average molecular weight)-3-oxypropionicacid (48 mg, 48 μmol) dissolved in anhydrous DMF (700 μL) andtriethylamine (13 μL, 95 μmol). After 30 min, a solution containingcytidine-5′-monophosphoryl-(5-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate)(30 mg, 48 μmol), water (400 μL) and triethylamine (13 μL, 95 μmol) wasadded. This solution was stirred 20 min at room temperature and thenchromatographed (C18 silica, gradient of methanol/water). Appropriatefractions were collected, concentrated, the residue dissolved in waterand freeze-dried to afford 40 mg (50% yield) of a white solid:R_(f)=0.36 (silica, IPA/H₂O/NH₄OH 7/2/1); ¹H NMR (D20, 500 MHz) δ 1.66(td, 1H, J=5.3), 2.50 (dd, 1H, J=13.2, 4.6), 2.64 (t, J=5.99, 3H) 3.43(d, J=9.58, 1H), 3.63 (m, 1H), 3.71 (s, 70H), 3.79 (m, obscured by 3.71peak), 3.82 (t, J=6.19, 1H) 3.88 (dd, J=11.8, 1.0, 1H), 3.95 (td, J=9.0,2.3, 1H), 3.98 (t, J=5.06, 1H), 4.12 (td, J=10.34, 4.66, 1H), 4.18 (d,J=10.36, 1H), 4.23 (d, J=4.85, 2H), 4.31 (t, J=4.64, 1H), 4.35 (t, 1H),6.00 (d, J=4.55, 1H), 6.13 (d, J=7.56, 1H), 7.98 (d, J=7.54, 1H). MS(MALDI), observe [M−H]; 1594.5, 1638.5, 1682.4, 1726.4, 1770.3, 1814.4,1858.2, 1881.5, 1903.5, 1947.3.

[1346] Preparation ofcytidine-5′-monophosphoryl-[5-(N-methoxy-polyoxyethylene-(10kDa)-oxycarboxamido)-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate].Cytidine-5′-monophosphoryl-(5-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate)(2.5 mg, 4 μmol) and water (180 μL) was added to a solution of(Methoxypolyoxyethylene-(10 kDa, average molecularweight)-oxycarbonyl-(N-oxybenzotriazole) ester (40 mg, 4 μmol) inanhydrous DMF (800 μL) containing triethylamine (1.1 μL, 8 μmol) and thereaction mixture stirred for 1 hr at room temperature. The reactionmixture was then diluted with water (8 mL) and was purified by reversedphase flash chromatography (C18 silica, gradient of methanol/water).Appropriate fractions were combined, concentrated, the residue dissolvedin water and freeze-dried yielding 20 mg (46% yield) of product as awhite solid: R_(f)=0.35 (silica, IPA/H₂O/NH₄OH 7/2/1); ¹H NMR (D20, 500MHz) δ 1.66 (td, 1H), 2.50 (dd, 1H), 2.64 (t, 3H) 3.55-3.7 (m, obscuredby 3.71 peak), 3.71 (s, 488H), 3.72-4.0 (m, obscured by 3.71 peak), 4.23(m, 3H), 4.31 (t, 1H), 4.35 (t, 1H), 6.00 (d, J=4.77, 1H), 6.12 (d,J=7.52, 1H), 7.98 (d, J=7.89, 1H). MS (MALDI), observe [M−CMP+Na];10780.

[1347] 2. Preparation of CMP-SA-PEG II This example sets forth thegeneral procedure for making CMP-SA-PEG, and specific procedures formaking CMP-SA-PEG (1 kDa) and CMP-SA-PEG (20 kDa).

[1348] General procedures PreparingCytidine-5′-monophosphoryl-(5-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate).Cytidine-5′-monophosphoryl-(5-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate(870 mg, 1.02 mmol) was dissolved in 25 mL of water and 5.5 mL of 40 wt% dimethylamine solution in H₂O was added. The reaction was stirred for1 hr and the excess dimethyl amine was then removed by rotaryevaporation. The aqueous solution was filtered through a C-18 silica gelcolumn and the column was washed with water. The eluants were combinedand lyophilized to afford 638 mg (93%) of a white solid. R_(f)=0.10(silica, IPA/H₂O/NH₄OH; 7/2/1). ¹H NMR (D20, 500 MHz) δ 1.66 (td, 1H,J=5.3), 2.50 (dd, 1H, J=13.2, 4.6), 3.43 (d, J=9.58, 1H), 3.63 (dd,J=11.9, 6.44, 1H), 3.88 (dd, J=11.8, 1.0, 1H), 3.95 (td, J=9.0, 2.3,1H), 4.10 (t, J=10.42, 1H), 4.12 (td, J=10.34, 4.66, 1H), 4.18 (d,J=10.36, 1H), 4.24 (m, 2H), 4.31 (t, J=4.64, 1H), 4.35 (t, 1H), 6.00 (d,J=4.37, 1H), 6.13 (d, J=7.71, 1H), 7.98 (d, J=7.64, 1H). MS (ES); calc.for C₂₁H₃₂N₅O₁₁P ([M−H]⁻), 629.47; found 627.9.

[1349] General procedures for Preparing CMP-SA-PEG usingmPEG-(p-nitrophenol)carbonate.Cytidine-5′-monophosphoryl-(5-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate)(175 mg, 0.259 mMol) was dissolved in a mixture of water, pH 8.5, andDMF or THF (in a ratio of 1:2). The mPEG-nitrophenol carbonate (2 to 20kDa mPEG's) (0.519 mMole) was added in several portions over 8 hr atroom temperature and the reaction mixture was stirred at roomtemperature for 3 days. When complete, water (40 ml) and 1.5 ml of NH₄OH(29% aqueous solution) were added. The yellow reaction mixture wasstirred for another 2 hr and then concentrated by rotary evaporation.The reaction mixture was then diluted with water (pH 8.5) to about 500ml volume and was purified by reversed phase flash chromatography(Biotage 40M, C18 silica column) with a gradient of methanol/water.Appropriate fractions were combined and concentrated to afford theproducts as white solids. R_(f) (silica; 1-propanol/water/29% NH₄OH;7/2/1); (2 kDa PEG)=0.31; (5 kDa PEG)=0.33; (10 kDa PEG)=0.36; (20 kDaPEG)=0.38 (TLC silica, IPA/H₂O/NH₄OH 7/2/1); MS (MALDI), observe[M−CMP+Na]; (2 kDa)=2460; (5 kDa)=5250; (10 kDa)=10700; (20 kDa)=22500.

[1350] Preparation ofCytidine-5′-monophosphoryl-[5-(N-fluorenylmethoxycarboxamido)-glycylamido-3,5-dideoXy-β-D-glycero-D-galacto-2-nonulopyranosuronate].Solium pyruvate (2.4 g, 218 mmol), HEPES buffer (0.25 M, pH 7.34) and1.0 g (22 mmol) of Fmoc-glycylmannosamide were mixed in a 150 mLpolycarbonate bottle. A neuraminic acid aldolase solution (19 mL, 600 U)was then added and the reaction mixture was incubated at 30° C. on anorbital shaker. After 23 hours, Thin layer chromatography (TLC)indicated that approximately 75% conversion to product had occurred. TheCTP (1.72 g, 33 mmol) and 0.1 M of MnCl₂ (6 mL) were then added to thereaction mixture. The pH was adjusted to 7.5 with 1 M NaOH (5.5 mL) anda solution containing CMP-neuraminic acid synthetase (Neisseria) wasadded (25 mL, 386 U). The reaction was complete after 24 hrs and thereaction mixture was chromatographed (C-18 silica, gradient from H₂O(100%) to 10% MeOH/H₂O). Appropriate fractions were recombined,concentrated and lyophilized to afford a white solid, R_(f)(IPA/H₂O/NH₄OH, 7/2/1)=0.52. ¹H NMR (D₂O, 500 MHz) δ 1.64 (dt, 1H,J=12.0, 6.0), 2.50 (dd, 1H, J=13.2, 4.9), 3.38 (d, J=9.67, 1H), 3.60(dd, J=11.65, 6.64, 1H), 3.79 (d, J=4.11, 1H), 3.87 (dd, J=12.24, 1.0,1H), 3.97 (m, 2H), 4.07 (td, J=10.75, 4.84, 1H), 4.17 (dd, J=10.68, 1.0,1H), 4.25 (s, 2H), 4.32 (t, J=4.4, 1H), 4.37 (t, J=5.8 1H), 4.6-4.7 (m,obscured by solvent peak), 5.95 (d, J=4, 1H), 6.03 (d, J=7.4, 1H),7.43-7.53 (m, 3H), 7.74 (m, 2H), 7.94 (q, J=7, 3H) MS (ES); calc. forC₃₅H₄₂N₅O₁₈P ([M−H]⁻), 850.7; found 850.8.

[1351] Preparation ofCytidine-5′-monophosphoryl-[5-(N-methoxypolyoxyethylene-(1kDa)-3-oxypropionamido)-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate].Methoxypolyoxyethylene-(1 kDa average molecularweight)-3-oxypropionate-N-succinimidyl ester (52 mg, 52 μmol) dissolvedin anhydrous DMF (450 mL) and triethylamine (33 μL, 238 μmol).Cytidine-5′-monophosphoryl-(5-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate)(30 mg, 48 μmol) was added as a solid. Water, pH 8 (330 μL) was addedand after 30 min, an additional 28 mg of NHS-activated PEG was added.After an additional 5 min, the reaction mixture was chromatographed(C-18 silica, gradient of methanol/water), and appropriate fractionswere concentrated to afford 32 mg (40% yield) of a white solid,R_(f)=0.31 (silica, IPA/H2O/NH₄OH 7/2/1); ¹H NMR (D₂O, 500 MHz) δ 1.66(td, 1H, J=5.3), 2.50 (dd, 1H, J=13.2, 4.6), 2.64 (t, J=5.99, 3H) 3.43(d, J=9.58, 1H), 3.63 (m, 1H), 3.71 (s, 70H), 3.79 (m, obscured by 3.71peak), 3.82 (t, J=6.19, 1H) 3.88 (dd, J=11.8, 1.0, 1H), 3.95 (td, J=9.0,2.3, 1H), 3.98 (t, J=5.06, 1H), 4.12 (td, J=10.34, 4.66, 1H), 4.18 (d,J=10.36, 1H), 4.23 (d, J=4.85, 2H), 4.31 (t, J=4.64, 1H), 4.35 (t, 1H),6.00 (d, J=4.55, 1H), 6.13 (d, J=7.56, 1H), 7.98 (d, J=7.54, 1H). MS(MALDI), observe [(M−CMP)−H]; 1506.4, 1550.4, 1594.5, 1638.5, 1682.4,1726.4, 1770.3, 1814.4, 1858.2.

[1352] Preparation ofCytidine-5′-monophosphoryl-{5-[N-(2,6-dimethoxypolyoxyethylene-(20kDa)-3oxypropionamidyl-lysylamido]-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate}.The 2,6-Di-[methoxypolyoxyethylene-(20 kDa average molecularweight)-3-oxypropionamidyl]-lysylamido-N-succinimidyl ester (367 mg, 9μmol) was dissolved in anhydrous THF (7 mL) and triethylamine (5 μL, 36μmol).Cytidine-5′-monophosphoryl-(5-glycylamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate)(30 mg, 48 μmol) was dissolved in 1.0 mL of water, and added to thereaction mixture. The reaction was stirred for 4 hours at roomtemperature and was then chromotographed (HPLC, Waters Xterra RP8,gradient from water/NH₄OH, 100% to 20% methanol/water/NH₄OH at 1 mL/min)to afford a white solid with a R_(t)=22.8 min. MS (MALDI), observe[(M−CMP)−H]; 43027.01 (40,000-45,500).

[1353] 3. Preparation of UDP-Gal-PEG.

[1354] This example sets forth the general procedure for makingUDP-Gal-PEG.

[1355] Methoxypolyoxyethylenepropionate N-hydroxysuccinimide ester(mPEG-SPA, MW 1,000) 348 mg in THF (0.5 mL) was added to a solution of25 mg of galactosamine-1-phosphate in 1 ml of water, followed by theaddition of 67 μL triethylamine. The resulting mixture was stirred atroom temperature for 17 hr. Concentration at reduce pressures provided acrude reaction mixture which was purified by chromatography (C-18silica, using a step gradient of 10%, 20%, 30%, 40% aqueous MeOH) toafford 90 mg (74%) of product after the appropriate fractions werecombined and concentrated to dryness. R_(f)=0.5 (silica,Propanol/H₂O/NH₄H 30/20/2); MS(MALDI), observed 1356, 1400, 1444, 1488,1532, 1576, 1620.

[1356][α-1-(Uridine-5′-diphosphoryl)]-2-deoxy-2-(methoxypolyoxyethylene-propionoylamido-1kDa)-α-D-galactosamine. The2-deoxy-2-(methoxy-polyoxyethylenepropionoylamido-1kDa)-α-1-monophosphate-D-galactosamine (58 mg) was dissolved in 6 mL ofDMF and 1.2 mL of pyridine. UMP-morpholidate (60 mg) was then added andthe resulting mixture was stirred at 70° C. for 48 hr. Afterconcentration, the residue was chromatographed (C18-silica, using a stepgradient of 10%, 20%, 30%, 40%, 50%, 80% MeOH) to yield 50 mg of productafter concentration of the appropriate fractions. R_(f)=0.54 (silica,propanol/H₂₀NH₄OH 30/20/2). MS(MALDI); Observed 1485, 1529, 1618, 1706.

[1357][α-1-(Uridine-5′-diphosphoryl)]-6-deoxy-6-(methoxypolyoxyethylene-amino-2kDa)-α-D-galactose.[α-1-(Uridine-5′-diphosphoryl)]-6-carboxaldehyde-α-D-galactose (10 mg)was disssolved in 2 mL of 25 mM sodium phosphate buffer (pH 6.0) andtreated with methoxypolyethyleneglycol amine (MW 2, 000, 70 mg) and then25 μL of 1M NaBH₃CN solution at 0° C. The resulting mixture was frozenat −20° C. for three days. The reaction mixture was chromatographed(HPLC, Water Xterra P8) using 0.015 M NH₄OH as mobile phase A and MeOHas mobile phase B as eluent at the speed of 1.0 mL/min. The product wascollected, an concentrated to yield a solid; R_(t)=9.4 minutes.R_(f)=0.27(silica, EtOH/H₂O 7/3).

[1358] [α-1-(Uridine-5′-diphosphoryl)]-6-amino-6-deoxy-α-D-galactose.Ammonium acetate 15 mg was added to a solution of[α-1-(Uridine-5′-diphosphoryl)]-6-carboxaldehyde-α-D-galactopyranoside(10 mg) in sodium phosphate buffer (pH 6.0). A solution of (25 μL) 1MNaBH₃CN was then added and the mixture was stirred for 24 hr. Thesolution was concentrated and the residue was chromotographed (sephadexG₁₀) to afford 10 mg of a white solid, R_(f)=0.62 (silica, EtOH/0.1MH₄Ac).

[1359][α-1-(Uridine-5′-diphosphoryl)]-6-deoxy-6-(methoxypolyoxyethylenepropionoylamido, ˜2 kDa)-α-D-galactopyranoside.[α-1-(Uridine-5′-diphosphoryl)]-6-amino-6-deoxy-α-D-galactopyranoside (5mg) was dissolved in 1 mL of H₂O. Thenmethoxypolyetheneglycolpropionoyl-NHS ester (MW ˜2,000, 66 mg) wasadded, followed by 4.6 μL triethylamine. The resulting mixture wasstirred at room temperature overnight, and then purified on HPLC (C-8silica) to afford the product, R_(t)=9.0 min.

[1360][α-1-(Uridine-5′-diphosphoryl)]-6-deoxy-6-(methoxypolyoxyethylenecarboxamido,˜2 kDa)-α-D-galactopyranoside.[α-1-(Uridine-5′-diphosphoryl)]-6-amino-6-deoxy-α-D-galactopyranoside(10 mg) was mixed with methoxypolyethyleneglycolcarboxy-HOBT (MW 2000,67 mg) in 1 mL of H₂O, followed by the addition ofEDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride 6.4 mgand 4.6 μL triethylamine. The resulting mixture was stirred at roomtemperature 24 hr. The mixture was chromatographed (C-8 silica) toafford the product.

[1361] 4. Preparation of UDP-GlcNAc-PEG

[1362] This example sets forth the general procedure for makingUDP-GlcNAc-PEG. On the left side of scheme 17, the protected amino sugardiphospho-nucleotide is oxidized to form an aldehyde at the 6-positionof the sugar. The aldehyde is converted to the corresponding primaryamine by formation and reduction of the Schiff base. The resultingadduct is contacted with the p-nitrophenol carbonate of m-PEG, whichreacts with the amine, binding the m-PEG to the saccharide nucleus viaan amide bond. On the right side of scheme 17 at the top, the protectedamino sugar diphospho-nucleotide is treated with a chemical oxidant toform a carboxyl group at the 6-carbon of the sugar nucleus. The carboxylgroup is activated and reacted with m-PEG amine, binding the m-PEG tothe saccharide nucleus via an amide bond. On the right side of scheme 17at the bottom the reactions are substantially similar to that on the topright, with the exception that the starting sugar nucleotide iscontacted with an oxidizing enzyme, such as a dehydrogenase, rather thana chemical oxidant.

[1363] 5. Preparation of UDP-GalNAc-PEG

[1364] This example (scheme 18) sets forth the general procedure formaking UDP-GalNAc-PEG. The reaction set forth above originates with asugar diphospho-nucleotide, in which R is either a hydroxyl 1 or aprotected amine 2. In step a, the starting sugar is treated with amixture of an oxidase and a catalase, converting the 6-postion of thesugar into an aldehyde moiety (3 and 4). In step c, the aldehyde isconverted to the corresponding amine (7 and 8) by formation andreduction of a Schiff base. In step e, the amine is optionally treatedwith an activated m-PEG derivative, thereby acylating the amine toproduce the corresponding m-PEG amide (11 and 13). Alternatively, instep f, the amine is contacted with an activated m-PEG species, such asa m-PEG active ester, thereby forming the corresponding m-PEG amide (12and 14). In step b, the starting material is also treated with acatalase and oxidase, completely oxidizing the hydroxymethyl moiety,forming a carboxyl group at the 6-position. In step d, the carboxylmoiety is activated and subsequently converted to a m-PEG adduct (9 and10) by reaction with a m-PEG amine intermediate. This is shown in scheme18.

[1365] The amino-sugar phosphate is contacted with a m-PEG N-hydroxysuccinimide active ester, thereby forming the correspondingsugar-PEG-amide. The amide is contacted with UMP-morpholidate to formthe corresponding active sugar diphospho-nucleotide.

[1366] 6. Synthesis of CMP-SA-Levulinate

[1367] This example sets forth the procedure for the synthesis ofCMP-SA-levulinate.

[1368] Preparation of 2-levulinamido-2-deoxy-D-mannopyranose.Isobutylchloroformate (100 μL, 0.77 mmol) was added dropwise to asolution of levulinic acid (86 μL, 0.84 mmol), anhydrous THF (3 mL) andtriethylamine (127 μL, 0.91 mmol). This solution was stirred for 3 hoursat room temperature and was then added dropwise to a solution containingD-mannosamine hydrochloride (151 mg, 0.7 mmol), triethylamine (127 μL,0.91 mmol), THF (2 mL) and water (2 mL). The reaction mixture wasstirred 15 hours and then concentrated to dryness by rotary evaporation.Chromatography (silica, step gradient of 5-15% MeOH/CH₂Cl₂) was used toisolate the product yielding 0.156 g (73% yield) of a white solid:R_(f)=0.41 (silica, CHCl₃/MeOH/water 6/4/1); ¹H NMR (D₂O, 500 MHz) δ2.23 (s, 3H), 2.24 (s, 3H), 2.57 (td, J=6.54, 3.68, 2H) 2.63 (t, J=6.71,2H), 2.86-2.90 (m, 4H), 3.42 (m, 1H), 3.53 (t, J=9.76, 1H), 3.64 (t,J=9.43, 1H), 3.80-3.91 (m, 4H), 4.04 (dd, J=9.79, 4.71, 1H), 4.31 (dd,J=4.63, 1.14, 1H), 4.45 (dd, J=4.16, 1.13, 1H), 5.02 (d, J=1.29, 1H),5.11 (s, J=1.30, 1H), MS (ES); calculated for C₁₁H₁₉NO₇, 277.27; found[M+1] 277.9.

[1369] Preparation of5-levulinamido-3,5-dideoxy-D-glycero-D-galacto-2-nonulopyranosuronate.Sodium pyruvate (0.616 g, 5.6 mmol) and N-acetylneuraminic acid aldolase(50 U) was added to a solution of 2-levulinamido-2-deoxy-D-mannopyranose(0.156 g, 0.56 mmol) in 0.1 M HEPES (pH 7.5). The reaction mixture washeated to 37° C. for 20 hours and after freezing. The reaction mixturewas then filtered through C18 silica, frozen and freeze-dried. The crudesolid was purified using flash chromatography (silica, first using10-40% MeOH/CH₂Cl₂ and then CH₂Cl₂/MeOH/H₂O 6/4/0.5). Appropriatefractions were combined and concentrated yielding 45 mg (80% yield) of awhite solid: R_(f)=0.15 (silica, CHCl₃/MeOH/water 6/4/1); ¹H NMR (D20,500 MHz) δ 1.82 (t, J=11.9, 1H), 2.21 (dd, J=13.76, 4.84, 1H), 2.23 (s,3H), 2.57 (app q, J=6.6, 2H), 2.86-2.95 (m, 2H), 3.15-3.18 (m, 1H),3.28-3.61 (complex, 1H), 3.60 (dd, J=11.91, 6.66, 1H), 3.75 (td, J=6.65,2.62, 1H), 3.84 (dd, J=11.89, 2.65, 1H), 3.88-4.01 (complex, 2H), 4.04(td, J=11.18, 4.67, 1H), MS (ES); calculated for C₁₄H₂₃NO₁₀, 365.33;found ([M−1]⁻), 363.97.

[1370] Preparation ofcytidine-5′-monophosphoryl-(5-levulinamido-3,5-dideoxy-β-D-glycero-D-galacto-2-nonulopyranosuronate).5-Levulinamido-3,5-dideoxy-D-glycero-D-galacto-2-nonulopyranosuronate(50 mg, 137 μmol) was dissolved in 2 mL of 100 mM HEPES pH 7.5 bufferand 1 M MnCl₂ (300 μL, 300 μmol) was added. CTP-2Na⁺ (79 mg, 1.5 μmol)was dissolved in 5 mL HEPES buffer and was added to the sugar. Thesialyltransferase/CMP-neuraminic acid synthetase fusion enzyme (11 U)was added and the reaction mixture stirred at room temperature for 45hours. The reaction mixture was filtered through a 10,000 MWCO filterand the filtrate, which contained the product of the reaction, was useddirectly without further purification: R_(f)=0.35 (silica,IPA/water/NH₄OH 7/2/1).

[1371] B. Glycoconiugation and GlycoPEGylation of Peptides

[1372] α-Protease Inhibitor (α-Antitrypsin)

[1373] 7. Sialylation of Recombinant Glycoproteins Antithrombin III,Fetuin and α1-Antitrypsin

[1374] This example sets forth the preparation of sialylated forms ofseveral recombinant peptides.

[1375] Sialylation of Recombinant Glycoproteins Using ST3Gal III.Several glycoproteins were examined for their ability to be sialylatedby recombinant rat ST3Gal III. For each of these glycoproteins,sialylation will be a valuable process step in the development of therespective glycoproteins as commercial products.

[1376] Reaction Conditions. Reaction conditions were as summarized inTable 11. The sialyltransferase reactions were carried out for 24 hourat a temperature between room temperature and 37°. The extent ofsialylation was established by determining the amount of ¹⁴C-NeuAcincorporated into glycoprotein-linked oligosaccharides. See Table 11 forthe reaction conditions for each protein. TABLE 11 Reaction conditions.CMP- Protein Protein ST ST/ NeuAc Total Conc. (mU/ Protein of ProteinSource (mg) (mg/ml) mL) (mU/mg) “cycle”¹ ATIII Genzyme 8.6 4.3 210 48cycle Transgenics ATIII Genzyme 860 403 53 12 cycle Transgenics Asialo-Sigma 0.4 105 20 13 10 mM fetuin asilao- PPL 0.4 0.5 20 20 20 mM AAAT

[1377] The results presented in Table 12 demonstrate that a remarkableextent of sialylation was achieved in every case, despite low levels ofenzyme used. Essentially, complete sialylation was obtained, based onthe estimate of available terminal galactose. Table 12 shows the relultsof the sialylation reactions. The amount of enzyme used per mg ofprotein (mU/mg) as a basis of comparison for the various studies. Inseveral of the examples shown, only 7-13 mU ST3Gal III per mg of proteinwas required to give essentially complete sialylation after 24 hours.TABLE 12 Analytical results Terminal NeuAc Gal¹ Incorp.² % Other ProteinSource mol/mol mol/mol Rxn³ characterization ATIII⁴ Genzyme 102 104 117None Transgenics ATIII⁴ Genzyme 102 1.3 108 SDS-gels: proteinTransgenics purity FACs: carbohydrate glycoforms Asialo- Sigma 802 905116 None fetuin asilao- PPL 7 7.0 100 SDS-gels: protein AAAT⁵ purity

[1378] These results are in marked contrast to those reported indetailed studies with bovine ST6Gal I where less than 50 mU/mg proteingave less than 50% sialylation, and 1070 mU/mg protein gaveapproximately 85-90% sialylation in 24 hours. Paulson et al. (1977) J.Biol. Chem. 252: 2363-2371; Paulson et al. (1978) J. Biol. Chem. 253:5617-5624. A study of rat α2,3 and α2,6 sialyltransferases by anothergroup revealed that complete sialylation of asialo-AGP required enzymeconcentrations of 150-250 mU/mg protein (Weinstein et al. (1982) J.Biol. Chem. 257: 13845-13853). These earlier studies taken togethersuggested that the ST6Gal I sialyltransferase requires greater than 50mU/mg and up to 150 mU/mg to achieve complete sialylation.

[1379] This Example demonstrates that sialylation of recombinantglycoproteins using the ST3 Gal III sialyltransferase required much lessenzyme than expected. For a one kilogram scale reaction, approximately7,000 units of the ST3Gal III sialyltransferase would be needed, insteadof 100,000-150,000 units that earlier studies indicated. Purification ofthese enzymes from natural sources is prohibitive, with yields of only1-10 units for a large scale preparation after 1-2 months work. Assumingthat both the ST6Gal I and ST3Gal III sialyltransferases are produced asrecombinant sialyltransferases, with equal levels of expression of thetwo enzymes being achieved, a fermentation scale 14-21 times greater (ormore) would be required for the ST6Gal I sialyltransferase relative tothe ST3Gal III sialyltransferase. For the ST6Gal I sialyltransferase,expression levels of 0.3 U/i in yeast has been reported (Borsig et al.(1995) Biochem. Biophys. Res. Commun. 210: 14-20). Expression levels of1000 U/liter of the ST3 Gal III sialyltransferase have been achieved inAspergillus niger. At current levels of expression 300-450,000 liters ofyeast fermentation would be required to produce sufficient enzyme forsialylation of 1 kg of glycoprotein using the ST6Gal Isialyltransferase. In contrast, less than 10 liter fermentation ofAspergillus niger would be required for sialylation of 1 kg ofglycoprotein using the ST3Gal III sialyltransferase. Thus, thefermentation capacity required to produce the ST3Gal IIIsialyltransferase for a large scale sialylation reaction would be 10-100fold less than that required for producing the ST6Gal I; the cost ofproducing the sialyltransferase would be reduced proportionately.

[1380] Cri-IgG Antibody

[1381] 8. Glyco-Remodeling of Cri-IgG1 Antibodies

[1382] This example sets forth the procedures for in vitro remodeling ofCri-IgG1 antibodies.

[1383] N-glycosylation at one conserved site at Asn 297 in the Fc domainof a monoclonal antibody can modulate its pharmacokinetic behavior andeffector functions (Dwek et al., 1995, J. Anat. 187:279-292; Boyd etal., 1995, Mol. Immunol. 32:1311-1318; Lund et al., 1995, FASEB J. 1995,9:115-119; Lund et al., 1996, J. Immunol. 157:4963-4969; Wright &Morrison, 1998, J. Immunol. 160:3393-3402; Flynn & Byrd, 2000, Curr.Opin. Oncol. 12:574-581). During cell culture fermentation or in certainpathological conditions, significant heterogeneity arises in theglycosylation pattern at this site. The resulting different patterns ofglycosylation on the Fc domain are characterized by complex biantennarystructures with zero, one, and two terminal galactose residues (G0, G1,and G2, respectively, see Table 13). The observed glycoform variations,such as the variation in terminal galactosylation, truncatedN-glycoforms and bisecting modification, have been shown to influencethe antibody's therapeutic properties, especially its ability to mediatetargeted cell killing through complement binding and activation (Boyd etal., 1995, supra; Wright & Morrison, 1998, supra, Mimura et al., 2000,Molec. Immunol. 37:697-706; Davies et al., 2001, Biotechnol. Bioeng.74:288-294).

[1384] In order to obtain different glycoforms of Cri-IgG1 antibodiesand test their Fc effector functions, Cri-IgG1 antibodies were trimmedback stepwise using exoglycosidases to generate glycoforms lackingsialic acid (G2, G1), glycoforms lacking sialic acid and galactose (G0),and glycoforms lacking sialic acid, galactose and N-acetyl glucosamine(M3N2F), as illustrated in Table 13. These molecules were subsequentlymodified using different glycosyltransferases and appropriate sugars.Modification conditions were developed that resulted in the conversionof the original antibody glycan structures into different glycoforms:M3N2, GnT-I-M3N2 (the M3M2 glycoform with a GlcNAc moiety added usingGnT-I), G0, Bisecting-G0 (the G0 moiety with a bisecting GlcNAc addedwith GnT-III), galactosylated bisecting-G0 (the bisecting-G0 glycoformwith terminal galactose moieties added), G2, mono-sialylated S1(α2,6)-G2(the G2 glycoform with one terminal sialic acid moiety added using(α2,6-sialyltransferase), S1(α2,3)-G2 (the G2 glycoform with oneterminal sialic acid moiety added using α2,3-sialyltransferase) anddisialylated S2(α2,3)-G2 (the G2 glycoform). After every glycoremodelingstep, the glycan structures were enzymatically released from theantibody protein and were analyzed by various methods, includingseparation by capillary electrophoresis, 2-AA HPLC profiling andMALDI-TOF mass spectrometry. TABLE 13 Abbreviations for glycoformstructures. Abbreviation Glycan Structure(s) M3N2(F)

G0

G1

G2

= fucose,

= GlcNAc,

= mannose,

= galactose

[1385] The materials ane methods used in these experiments are nowdescribed.

[1386] The Cri-IgG1 Monoclonal Antibody. The Cri-IgG1 antibody wasobtained from R. Jefferies, MRC Center for Immune Regulation, TheMedical School, University of Birmingham, UK. The antibody is anon-recombinant antibody, and is isolated from a human myeloma. Theantibody was prepared using three methods. In the first method, referredto as “DEAE,” the antibody was isolated under relatively mild conditionsusing a DEAE ion exchange column. In the second method, referred to as“SPA,” the antibody was purified on a protein A column (Staphylococcusaureus protein A) with a low pH elution step. In the third method,referred to as “Fc,” the antibody was treated with a protease so thatonly the Fc portion of the antibody remained and the antigen bindingdomains were removed. These methods for antibody purification are wellknown to those of skill in the art and are not repeated in detail here.

[1387] Affinity purification of remodeled antibodies. Antibody, modifiedeither by exoglycosidase or glycosyltransferase, was affinity purifiedon a ProA-sepharose 4-fast flow column (Amersham Bioscience, ArlingtonHeights, Ill.), eluted with 0.1 M glycine-HCl buffer, pH 2.7, andimmediately neutralized with 1 M Tris, pH 9.5. The eluates werebuffer-exchanged using a NAP-10 column (Amersham Bioscience, ArlingtonHeights, Ill.) to an appropriate buffer for the next step ofglycosylation, such as 100 mM MES, pH 6.5 or 50 mM Tris-HCl, pH 7.2. Theremodeled final products were dialyzed extensively against PBS at 4° C.in Tube-O-Dialyzers™ (Chemicon International, Temecula, Calif.) with aMWCO of 8 kDa.

[1388] In vitro glycosidase treatment of Cri-antibodies. Antibody wasbuffer-exchanged into 50 mM Na phosphate/Citrate, pH 6.0 using NAP-10column (Amersham Bioscience, Arlington Heights, Ill.). In vitro trimmingback of sugar moieties was carried out stepwise, by contacting theantibody (5 mg/mL) with 20 mU/mg protein neuraminidase at 37° C.overnight (to remove terminal sialic acid moieties), 20 mU/mg proteinβ-galactosidase at 37° C., overnight (to remove terminal galactosemoieties to result in the G0 glycoform), and/or 2 U/mgβ-N-acetylhexosaminidase (from Jack Bean, Seikagaku, Tokyo, Japan) at37° C., overnight (to remove terminal N-acetyl glucosamine to result inthe M3N2 glycoform). The samples were affinity purified as describedabove.

[1389] In vitro glvcosylation of Cri-antibodies. In vitro GnT1modification was performed using 1 mg/ml of the M3N2 glycoform antibodyas the substrate, and 25 mU/mg of recombinant humanβ1,2-mannosyl-UDP-N-acetylglucosaminosyltransferase in a buffer of 100mM MES, pH 6.5, 5 mM MnCl₂, 5 mM UDP-GlcNAc, and 0.02% NaN₃ at 32° C.for 24 hr. An aliquot was removed for glycan analysis, and the resultingproducts were affinity purified as described above.

[1390] In vitro modification of the bisecting-glycoform was carried outusing 1 mg/ml of the M3N2 glycoform antibody as the substrate and 25mU/mg of β1,2-recombinant humanmannosyl-UDP-N-acetylglucosaminosyltransferase I, 25 mU/mg ofβ1,2-recombinant human mannosyl-UDP-N-acetylglucosaminosyltransferase IIand 3.5 mU/mg of β1,4-recombinant mousemannosyl-UDP-N-acetylglucosaminosyltransferase III in a buffer of 100 mMMES pH 6.5, 10 mM MnCl₂, 5 mM UDP-GlcNAc, and 0.02% NaN₃ at 32° C. for24 hrs. An aliquot was removed for glycan analysis, and the remainingproduct was affinity purified as described above.

[1391] In vitro galactosylation was performed using G0 glycoformantibody or bisecting glycoform antibody by contacting the antibody with0.6 U/mg recombinant bovine milk β1,4 galactosyltransferase in a bufferof 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM UDP-galactose, 5 mM MnCl₂,at 32° C. for 24 hrs. An aliquot was removed for glycan analysis, andthe remaining products were affinity purified as described above.

[1392] In vitro sialylation was carried out using the G2 glycoformantibody (1 mg/mL) by contacting it with 0.1 U/mg ST3Gal3 or 0.1 U/mgST6Gal 1, 5 mM CMP-sialic acid, at 32° C. for 24 hr in a buffer of 50 mMTris pH 7.4, 150 mM NaCl, and 3 mM CMP-SA. An aliquot was removed forglycan analysis, and the remaining products were affinity purified asdescribed above.

[1393] Glycan Analysis:

[1394] Capillary Electrophoresis with Laser Induced FluorescenceDectection. Buffer components and nucleotide sugars were removed from analiquot of the glycoremodeled antibody by dilution and concentration ina Microcon™ YM-30 microconcentrator (Millipore, Bedford, Mass.).N-linked oligosaccharides were released from the protein by contactingit with PNGase F (Prozyme, San Leandro, Calif.) using the methodologyprovided by the manufacturer. In brief, the sample was denatured in thebuffer of 50 mM sodium phosphate pH 7.5, 0.1% SDS, and 50 mMβ-mercaptoethanol for 10 min at 100° C. TX100 was then added to 0.75%(v/v) as well as 10U PNGaseF/200 μg protein. After 3 hours incubation at37° C., the protein was ethanol precipitated and the supernatant wasdried down. The released free oligosaccharides were then labeled with8-aminopyrene-1,3,6-trisulfonic acid and analyzed by capillaryelectrophoresis with a carbohydrate labeling and analysis kit fromBeckman-Coulter, Inc. (Fullerton, Calif.), as indicated by themanufacturer (see also, Ma and Nashabeh, 1999, Anal. Chem.71:5185-5192).

[1395] Capillary electrophoresis (CE) was carried out in an eCAP™ N—CHOcoated Capillary (50 μm I.D., length to detector 40 cm; Beckman-Coulter,Inc., Fullerton, Calif.), using a P/ACE™ MDQ Glycoprotein System(Beckman-Coulter, Inc. Fullerton, Calif.) with Laser InducedFluorescence Detector (Beckman-Coulter, Inc. Fullerton, Calif.). Sampleswere introduced into the cartridge by 20 psi pressure for 10 sec. andseparated under 25 kV with reverse polarity for 20 min. Cartridgetemperature was kept at 20° C. The electropherogram was generated bylaser-induced fluorescence detection at an excitation wavelength of 488nm and an emission wavelength of 520 nm.

[1396] Carbohydrate standards (Calbiochem®, EMD Biosciences, Inc., SanDiego, Calif.), including M3N2 (N-linked trimannosyl core without corefucose), G0 (N-linked oligosaccharide, asialo, agalacto, biantennarywith core fucose), G2 (N-linked oligosaccharide, asialo, biantennarywith core fucose), and G2 without fucose, S1-G2 (mono-sialylated,galactosylated biantennary oligosaccharide without core fucose) andS2-G2 (di-sialylated, galactosylated biantennary oligosaccharide withoutcore fucose), (from Glyko, see, ProZyme, San Leandro, Calif.), M3N2F(N-linked trimannosyl core with core fucose) and NGA2F (N-linkedoligosaccharide asialo, agalacto, biantennary with core fucose and withbisecting GlcNAc) were labeled with 1-aminopyrene-3,6,8-trisulfonate(APTS, Beckman-Coulter, Inc. Fullerton, Calif.) and used to identify thedistribution of glycans released from the antibody.

[1397] 2-AA HPLC. PNGaseF released glycans were labeled with 2-AA(2-anthranilic acid) according to the method described by Anumula andDhume with slight modifications (1998, Glycobiology 8:685-694).Reductively-aminated N-glycans were analyzed using a Shodex AsahipakNH2P-50 4D amino column (4.6 mm×150 mm) (Showa Denko K.K., Tokyo,Japan). The two solvents used for the separation are A) 2% acetic acidand 1% tetrahydrofuran in acetonitrile and B) 5% acetic acid, 3%triethylamine and 1% tetrahydrofuran in water.

[1398] To separate neutral 2AA-labeled glycans, the column was elutedisocratically with 70% A for 5 minutes, followed by a linear gradientover a period of 60 minutes going from 70% to 50% B, followed by a steepgradient over a period of 5 minutes going from 50% to 5% B and a finalisocratic elution with 5% B for 10 minutes. Eluted peaks were detectedusing fluorescence detection with an excitation at 230 nm and detectionwavelength at 420 nm. In this gradient condition, the G0 glycoform willelute at about 30.5 minutes, the G1 glycoform at about 34.0 minutes andthe G2 glycoform at about 37.0 minutes. Under these conditions, thepresence of fucose does not change the elution time.

[1399] To separate anionic 2AA-labeled glycans, the column was elutedisocratically with 70% A for 2.5 minutes, followed by a linear gradientover a period of 97.5 min going from 70% to 5% A and a final isocraticelution with 5% A for 15 minutes. Eluted peaks were detected usingfluorescence detection with excitation at 230 nm and detection at 420nm. In this gradient, neutral glycans are expected to elute between18.00-29.00 minutes, glycans with one charge elute between 30.00-40.00minutes, glycans with two charges elute between 43.00-52.00 minutes,glycans with three charges elute between 54.00-63.00 minutes, andglycans with four charges elute between 65.00-74.00 minutes.

[1400] MALDI analysis of reductively-aminated N-glycans. A small aliquotof the PNGase-released N-glycans that were labeled with 2-anthranilicacid (2AA) were then dialyzed for 45 minutes on a MF-Millipore membranefilter (0.025 μm pore, 47 mm dia.), which was floating on water. Thedialyzed aliquot was dried in a Speedvac™ (ThermoSavant, Holbrook,N.Y.), redissolved in a small amount of water, and mixed with a solutionof 2,5-dihydroxybenzoic acid (10 g/L) dissolved in water/acetonitrile(50:50).

[1401] The mixture was dried onto a MALDI target and analyzed using anApplied Biosystems DE-Pro mass spectrometer (Applied Biosystems, Inc.,Foster City, Calif.) operated in the linear/negative-ion mode.Oligosaccharide structures were assigned based on the observedmass-to-charge ratio and literature precedence. No attempt was made tofully characterize isobaric structures.

[1402] SDS-PAGE. To determine the stability of the glycoremodeledantibody, all the samples were analyzed by SDS-PAGE. The final productsof the samples were run under non-reducing conditions using 8-16%Tris-glycine gel (Invitrogen, Carlsbad, Calif.). Bovine serum albuminwas run under reducing condition as quantitative standards. The gel wasstained with GelCode Blue Stain Reagent (Pierce Chemical Co., Rockford,Ill.) for visualization.

[1403] The results of the experiments are now described.

[1404] Native glycoforms of Cri expressed in human myeloma cells.Cri-IgG1 antibody purified from the serum of a patient having multiplemyeloma contains variable glycoforms. FIGS. 97A-97C shows the HPLCprofiles of glycans enzymatically released from Cri-IgG1 antibody. FIGS.98A-98C shows the MALDI profiles of glycans enzymatically released fromCri-IgG1 antibody expressed in human mycloma cells. The major forms areunder-galactosylated G0, G1, while G2 and sialylated structures arerelatively minor (Table 14 and FIG. 97C). To test the impact of modifiedglycans on the therapeutic properties of the monoclonal antibody,Cri-IgG1 antibody was modified by performing in vitro exoglycosidasestrimming and in vitro glycosylation remodeling to generate differentglycoforms of this antibody. TABLE 14 Relative amount of differentglycoforms of human myeloma cell-expressed Cri-IgG1 separated by HPLCwas calculated from the areas of individual peaks. Criantibodies S1G2 G2G1 G0 DEAE 45.04 54.96 SPA 6 3.17 48.25 51.75 Fc 51.41 38.83

[1405] Initially, optimization of each step in exoglycosidases trimmingand glycosylation was performed at small scale (100 μg of each).

[1406] Trimannosyl core glycoform of Cri-IgG1 Antibody (M3N2). M3N2 wascreated by stepwise treatment of glycosidases, including neuraminidase,β1,4-galactosidase and β1-2, 3, 4, 6 N-acetylhexosaminidase. To assessthe removal of terminal galactose and GlcNAc on the glycoremodeledCri-IgG1 antibody samples, a quantitative capillary electrophoresis (CE)method was used. The glycans were enzymatically released from theglycoremodeled antibody with PNGase F and were derivatized with8-aminopyrene-1,3,6-trisulfonic acid (APTS) at the reducing terminus.The resulting products were analyzed by CE with on-column laser-inducedfluorescence detection (LIF) (Ma & Nashabeh, 1999, supra). Since theseparation of the glycans is based on the differences in hydrodynamicsize, the APTS labeled glycans migrate in order of increasing size(M3N2<M3N2F<G0<G1<G2).

[1407] FIGS. 99A-99D show the electropherograms indicating the glycansreleased from glycoremodeled Cri-IgG1 antibody as well as glycanstandards derivatized with APTS (FIG. 99A). The glycoforms wereidentified by comparing their electrophoretic mobilities to thestandards. The relative amount of each glycan species was calculatedfrom the relative area percentage of each indicated peak, and theresults are presented in Table 15. The M3N2F glycoform represents 91% ofthe glycans of DEAE-Cri, 80% of the glycans of SPA-Cri, and 100% of theglycans of Fc-Cri. Incomplete removal of GlcNAc moiety resulting in theGnT-I-M3N2F glycoform (see, Table 15) was observed in the glycanstructures from DEAE-Cri (8.6%) and SPA-Cri (˜20%). GlycoformGnT-I-M3N2F is the M3N2F glycoform with one additional GlcNAc, such aswould be added by GnT-I. TABLE 15 The areas of individual peaks from CEprofile in FIG. 99 were calculated, and relative amounts of the M3N2Fand GnT-I-M3N2F glycoforms were determined. M3N2F GnT-I-M3N2F RT (min.)% RT (min.) % DEAE 10.133 91.4 10.842 8.6 SPA 10.133 80.01 10.842 19.99Fc 10.133 100 10.842 0

[1408] Degalactosylated glycoform (G0). Cri-IgG1 antibody with G0glycoforms was obtained by stepwise treatment the native Cri-IgG1antibody with neuraminidase and β1,4-galactosidase in for 24 hours foreach reaction. The glycans released from the glycoremodeled antibodywere analyzed by CE, HPLC and MALDI. FIG. 100A shows the CE profile ofthe released glycans. In all three samples, only one peak was observedwhich was designated as the G0 glycoform based on comparison with thestandards (FIG. 100A and Table 16). TABLE 16 The relative amount of theG0 glycoform of Cri-IgG1 determined by CE and HPLC. CE HPLC RT (min.) %RT (min.) % DEAE 11.408 100.0 31.194 100.0 SPA 11.408 100.0 31.194 100.0Fc 11.408 100.0 31.194 100.0

[1409] In addition to the glycan analysis provided by CE, a quantitativeHPLC method was also used to determine the percent of the G0 glycoformrepresented by remodeled glycans of the Cri-IgG1 antibody. The glycandistribution on the glycoremodeled antibody was monitored byenzymatically releasing the glycans with PNGase F and derivatizing thereleased products with 2-anthranilic acid (2-AA) at the reducingterminus. The derivatized mixture was separated by HPLC on a ShodexAsahipak NH2P-50 4D column with fluorescence detection. FIGS. 101A-101Cshow the chromatograms obtained from the released glycans. HPLC resultsconfirmed CE analysis, as only one major peak was found in all threesamples. In agreement with CE and HPLC data, MALDI analysis also showedalmost complete glycoremodeling to the G0 glycoform (FIGS. 102A-102C).

[1410] Fully galactosylated G2 glycoform (G2). Cri-IgG antibodies weretreated with neuraminidase to yield asialo-glycoforms which were alsounder galactosylated. These asialoglycoforms were then treated with 0.6U/ml of bovine β1,4 galactosyltransferase and a galactose donor moleculeto glycoremodel the antibody to have the G2 glycoform.

[1411] The extent of terminal galactosylation was determined by glycananalysis. Only one major peak was observed in both CE and HPLC profiles(FIGS. 103A-103C and FIGS. 104A-104C). This peak corresponds to the G2glycoform in each case. Calculation of the percent total peak areashowed almost complete (˜90%) conversion to the G2 from the undergalactosylated glycoforms of the original samples (see, Table 14). Theseresults are summarized in Table 17. MALDI analysis of the glycansfurther supported the almost to complete glycoremodeling to the G2glycoform in all of the samples (FIGS. 105A-105C). TABLE 17 Relativeamount of G2 glycoform of remodeled Cri-IgI1 antibody determined bypercent total peak area in CE and HPLC analysis. CE HPLC RT (min.) % RT(min.) % DEAE 12.94 90 31.194 100 SPA 12.94 92 31.194 90 Fc 12.94 8431.194 89

[1412] GnT-I-glycoform (GnT-I-M3N2). The M3N2 glycoform Cri-IgG antibodywas glycoremodeled to the GnT-I-M3N2 glycoform by adding one GlcNAcmoiety to the molecule. The molecule was contacted with 25 mU GnT-I/mgantibody and an appropriate GlcNAc donor molecule. CE, HPLC and MALDIanalysis of released glycans (FIGS. 106A-106D, FIGS. 107A-107C and FIGS.108A-108C, respectively) indicated that the original M3N2F glycoform wascompletely remodeled. However, only 40-60% of the modified structureswere the GnT-I-M3N2 glycoform, and about 30% were the G0 glycoform. Thepresence of the G0 glycoform may be the result of incomplete GlcNActrimming when making the original M3N2 form.

[1413] Bisecting glycoform (NGA2F). The M3N2 glycoform Cri-IgG antibodywas glycoremodeled to the NGA2F glycoform by contacting it with acombination the three transferases, GnT-I, GnT-II and GnT-III, and anappropriate N-acetylglucosamine donor molecule. The reaction wascompleted in 24 hours. To determine the extent to which thebisecting-GlcNAc moiety was added to the glycans, CE analysis was usedto determine the glycoforms present on the glycoremodeled antibody.

[1414] FIGS. 109A-109D shows the electropherograms obtained from CEanalysis of the glycans released from glycoremodeled Cri-IgG1 antibody.Four peaks appeared after remodeling. A major peak migrated at the sameretention time as the NGA2F standard glycoform. The three other minorpeaks are likely to be the incompletely remodeled glycans. Forcomparison, a quantitative HPLC method was also used, where the 2-AAlabeled glycans eluted in order of increasing size (Gn1<G0<NGA2F). Asshown in FIGS. 110A-110C, similar results were obtained from the CEanalysis of the glycans. No M3N2F was found using either the CE or HPLCanalysis. NGA2F glycans were the major peaks I both CE and HPLCanalysis. The Gn1 and G0 glycans still remaining in the sample likelyare the result of incomplete modification. Most of the original M3N2Fglycoforms were remodeled by three GlcNAc moieties to the NGA2Fglycoform (60˜70%), about 15˜18% were remodeled by the addition of twoGlcNAc moieties to the G0 glycoform, and only small amount (7%) wereremodeled by the addition of only one GlcNAc moiety. MALDI-MS analysisof the released glycans (FIGS. 11A-11C) shows peaks of glycoforms withone, two or three terminal GlcNAc moieties, in agreement with CE andHPLC analysis (FIGS. 109 and 110). The relative amount of each glycanspecies was calculated from the relative area percentage of eachindicated peak, and is summarized in Table 18. TABLE 18 Relative amountsof different glycoforms from GnT-I, II, and III remodeled Cri-IgG1, asdetermined by CE and HPLC. Retention % Peak Area (min.) DEAE SPA Fc CEPeak 1 10.238 6.39 6.89 7.98 Peak 2 10.775 15.82 14.29 17.9 Peak 311.325 14.14 8.87 15.69 Bisec. 11.625 63.65 70.04 58.43 HPLC Peak 121.117 37.4 15.02 14 Peak 2 26.817 12.9 14.24 10.15 Peak 3 31.224 14.782.11 30.2 Bisec. 32.078 34.93 68.63 45.64

[1415] Galactosylated Bisecting (Gal-NGA2F) glycoforms. NGA2F glycoformsof Cri-IgG1 antibodies were glycoremodeled with bovineβ1,4-galactosyltransferase and an appropriate galactose donor. Theterminal galactose moieties were added using 0.6 U/ml of β1,4galactosyltransferase. FIGS. 112A-112D shows the electropherogramsobtained using the 2-AA HPLC method. In brief, the glycoformsterminating in GalNAc were almost 100% galactosylated. Comparing FIG.112A to FIG. 112B for DEAE Cri-IgG1, and FIG. 112C to FIG. 112D for FcCri-IgG1, the 2-AA HPLC profile of GnT-I, II and III modified glycans(FIGS. 112A and 112C) is modified by GalT1 so that all of the glycanpeakes were shifted to elute later due to the size increase from addedgalactose moieties (FIGS. 112B and 112D). These results were furtherconfirmed by MALDI-MS analysis.

[1416] Sialylated (S2G2) glycoforms of Cri-IgG1. The glycoremodeled G2glycoforms of Cri-IgG1 antibody were further remodeled using bothST3Gal3 and ST6Gal1. FIGS. 113A-113C shows the HPLC profile of the G2glycoforms remodeled with ST3Gal3. Most of the G2 glycoforms wereconverted into S2G2 glycoforms (the G2 glycoform with 2 additionalterminal sialic acid moieties; ˜70%, see, Table 19), and only smallamounts were the S1G2 glycoform (the G2 glycoform with 1 additionalterminal sialic acid moiety; <25%, see Table 19). These results werefurther confirmed in the MALDI analysis shown in FIGS. 114A-114C. MALDIdata also shows that all the G2 glycoforms were sialylated to eitherS2G2 or S1G2 glycoforms. TABLE 19 Relative amounts of differentglycoforms from ST3Gal3 remodeled Cri-IgG1 as determined by HPLC. RT(min.) DEAE SPA Fc S1G2 36.7 25.6 24.83 23.39 46.9 4.12 6.83 S2G2 49.458.93 50.68 61.88 52.19 9.1 7.56 6.07

[1417] By comparison, ST6Gal1 remodeling of the G0 glycoform did notreach the level of completion found with ST3Gal3 remodeling. FIGS.115A-115D and FIGS. 116A-116C show the results obtained from CE and HPLCanalysis, respectively. No S2G2 glycoforms were seen in any of theglycoremodeled samples. However, all of the G2 glycoforms were convertedinto S1-G2. Analysis from MALDI-MS also supports these data (FIGS.117A-117C).

[1418] Stability of remodeled glycans of Cri-IgG1. Lastly, the stabilityof the Cri-IgG1 glycans remodeled by exoglycosidase treatment andglycosylation was investigated. Each glycoremodeled Cri-IgG1 antibodywas stored at 4° C., and was checked by SDS-PAGE for degradation at twoweeks after remodeling. As shown in FIGS. 118A-118E, the remodeled DEAEand SPA antibodies both retained a molecular weight of about 150 kDa,indicating little to no degradation, regardless of the kind ofglycoremodeling performed. The Fc Cri-IgG1 antibody retained a molecularweight of about 38 kDa, also indicating little to no degradation,regardless of the kind of remodeling performed.

[1419] Effector Function Bioassay of Remodeled Cri-IgG1 antibodies. Theeffector function bioassay was derived from the procedure of Mimura etal. (2000, Molecular Immunology 37:697-706). The IC₅₀ of the glycoformsof Cri-IgG1 antibody was determined by inhibition of the superoxideresponse of U937 cells elicited by red blood cells sensitized withnative anti-NIP antibody.

[1420] Monocytic U937 cells were cultured in the presence of 1000units/mL interferon gamma for 2 days to induce the differentiation ofthe cells and their capacity to generate superoxide. The cells were thenwashed and resuspended at 2×10⁶ cells/mL in Hanks balanced salt solutionwithout phenol red and containing 20 mM HEPES pH 7.4 and 0.15 mM BSA.The red blood cells were sensitized with anti-NIP(5-iodo-4-hydroxy-3-nitrophenacetyl) antibody, in the absence orpresence of the various glycoforms of Cri-IgG1 antibody, with incubationat 37° C. for 30 minutes. The cells were then washed three times withPBS and resuspended at 2.5×10⁷ cells/mL in HBSS-BSA. The U937 cells (100μl, 2×10⁶ cells/mL) were added to plastic tubes and lucigenin (20 μl,2.5 mM) was added to the tubes. The tubes were warmed in a 37° C. waterbath for 5 minutes. The sensitized red blood cells (80 μl, 2.5×10⁷/mL)were then added to the tubes. Superoxide anion production was measuredby lucigenin-enhanced chemiluminescence at 37° C. over a 30 minuteperiod using a Berthold LV953 luminometer (Berthold Australia Pty Ltd,Bundoora, Australia).

[1421] The G0 and M3N2 glycoforms Cri-IgG1 antibody had relativeinhibitory values of 92% and 85%, respectively, as compared with thenative antibody. However, the native CRI-IgG1 antibody lacked corefucose. Shields et al. (2002, J. Biol. Chem. 277:26733-26740) suggeststhat the lack of core fucose will improve inhibitory values 10 fold.Based on these results, it is anticipated that inhibitory values of thegalactosylated-bisecting-G0 glycoform will be greater than thebisecting-G0 glycoform, which in turn will be much greater than the G2glycoform, which in turn will be approximately equal to thedisialylated-G2 glycoform and the monosialylated-G2 glycoform, which inturn will be greater than the native antibody glycoform, which in turnwill be greater than the G0 glycoform, which in turn will be greaterthan the M3N2 glycoform.

[1422] Complement Receptor-1

[1423] 9. Sialylation and Fucosylation of TP10

[1424] This example sets forth the preparation of TP10 with sialyl LewisX moieties and analysis of enhanced biological activity.

[1425] Interrupting blood flow to the brain, even for a short time, cantrigger inflammatory events within the cerebral microvasculature thatcan exacerbrate cerebral tissue damage. The tissue damage that accruesis amplified by activation of both inflammation and coagulationcascades. In a murine model of stroke, increased expression ofP-selectin and ICAM-1 promotes leukocyte recruitment. sCR1 isrecombinant form of the extracellular domain of Complement Receptor-1(CR-1). sCR-1 is a potent inhibitor of complement activation.sCR1sLe^(X) (CD20) is an alternately glycosylated form of sCR1 that isalternately glycosylated to display sialylated Lewis X antigen.Previously, sCR-1sLeX that was expressed and glycosylated in vivo inengineered Lec II CHO cells was found to correctly localize to ischemiccerebral microvessels and C1q-expressing neurons, thus inhibitingneutrophil and platelet accumulation and reducing cerebral infarctvolumes (Huang et al., 1999, Science 285:595-599). In the presentexample, sCR1sLe^(X) which was prepared in vitro by remodeling ofglycans, exhibited enhanced biological activity similar to that ofsCRsLe^(X) glycosylated in vivo.

[1426] The TP10 peptide was expressed in DUK B 11 CHO cells. This CHOcell line produces the TP10 peptide with the typical CHO cellglycosylation, with many but not all glycans capped with sialic acid.

[1427] Sialylation of 66 mg of TP10. TP10 (2.5 mg/mL), CMPSA (5 mM), andST3Gal3 (0.1 U/mL) were incubated at 32° C. in 50 mM Tris, 0.15M NaCl,0.05% sodium azide, pH 7.2 for 48 hours. Radiolabelled CMP sialic acidwas added to a small aliquot to monitor incorporation. TP10 wasseparated from nucleotide sugar by SEC HPLC. Samples analyzed at 24hours and 48 hours demonstrated that the reaction was completed after 24hours. The reaction mixture was then frozen. The reaction products weresubjected to Fluorophore Assisted Carbohydrate Electrophoresis (FACE®;Glyko, Inc, Novato Calif.) analysis (FIG. 119).

[1428] Pharmacokinetic studies. Rats were purchased with a jugular veincannula. 10 mg/kg of either the pre-sialylation or post-sialylation TP10peptide was given by tail vein injection to three rats for eachtreatment (n=3). Fourteen blood samples were taken from 0 to 50 hours.The concentration in the blood of post-sialylation TP10 peptide washigher than that of pre-sialylation TP10 at every time point past 0 hour(FIG. 120). Sialic acid addition doubled the area under the plasmaconcentration-time curve (AUC) of the pharmacokinetic curve as comparedto the starting material (FIG. 121).

[1429] Fucosylation of sialylated TP10. 10 mL (25 mg TP10) of the abovesialylation mix was thawed, and GDP-fucose was added to 5 mM, MnCl₂ to 5mM, and FTVI (fucosyltransferase VI) to 0.05 U/mL. The reaction wasincubated at 32° C. for 48 hours. The reaction products were subjectedto Fluorophore Assisted Carbohydrate Electrophoresis (FACE®; Glyko, Inc,Novato Calif.) analysis (FIG. 122). To a small aliquot, radiolabelledGDP-fucose was added to monitor incorporation. TP10 was separated fromnucleotide sugar by SEC HPLC. Samples analyzed at 24 hours and 48 hoursdemonstrated that the reaction was completed at 24 hours. An in vitroassay measuring binding to E-selectin indicate that fucose addition canproduce a biologically-active E-selectin ligand (FIG. 123).

[1430] Enbrel™

[1431] 10. GlycoPEGylation of an antibody Enbrel™ This example setsforth the procedures to PEGylate the O-linked glycans of an antibodymolecule. Here, Enbrel™ is used as an example, however one of skill inthe art will appreciate that this procedure can be used with manyantibody molecules.

[1432] Preparation of Enbrel™-SA-PEG (10 kDa). Enbrel™(TNF-receptor-IgG₁-chimera), either with the O-linked glycans sialylatedprior to PEGylation or not, is dissolved at 2.5 mg/mL in 50 mM Tris-HCl,0.15 M NaCl, 5 mM MnCl₂, 0.05% NaN₃, pH 7.2. The solution is incubatedwith 5 mM UDP-galactose and 0.1 U/mL of galactosyltransferase at 32° C.for 2 days to cap the undergalactosylated glycans with galactose. Tomonitor the incorporation of galactose, a small aliquot of the reactionhas ¹⁴C-galactose-UDP ligand added; the label incorporated into thepeptide is separated from the free label by gel filtration on a TosoHaas G2000SW analytical column in methanol and water. The radioactivelabel incorporation into the peptide is quantitated using an in-lineradiation detector.

[1433] When the reaction is complete, the solution is incubated with 1mM CMP-sialic acid-linker-PEG (10 kDa) and 0.1 U/mL of ST3Gal3 at 32° C.for 2 days. To monitor the incorporation of sialic acid-linker-PEG, thepeptide is separated by gel filtration on a Toso Haas G3000SW analyticalcolumn using PBS buffer (pH 7.1). When the reaction is complete, thereaction mixture is purified using a Toso Haas TSK-Gel-3000 preparativecolumn using PBS buffer (pH 7.1) and collecting fractions based on UVabsorption. The fractions containing product are combined, concentrated,buffer exchanged and then freeze-dried. The product of the reaction isanalyzed using SDS-PAGE and IEF analysis according to the procedures andreagents supplied by Invitrogen. Samples are dialyzed against water andanalyzed by MALDI-TOF MS.

[1434] Erythropoietin (EPO)

[1435] 11. Addition of GlcNAc to EPO

[1436] This example sets forth the addition of a GlcNAc residue on to atri-mannosyl core.

[1437] Addition of GlcNAc to EPO. EPO was expressed in SF-9 insect cellsand purified (Protein Sciences, Meriden, Conn.). A 100% conversion fromthe tri-mannosyl glycoform of Epo to the “tri-mannosyl core+2 GlcNAc”(Peak 1, P1 in FIG. 124) was achieved in 24 hours of incubation at 32°C. with 100 mU/ml of GlcNAcT-I and 100 mU/ml of GlcNAcT-II in thefollowing reaction final concentrations:

[1438] 100 mM MES pH 6.5, or 100 mM Tris pH 7.5

[1439] 5 mM UDP-GlcNAc

[1440] 20 mM MnCl2

[1441] 100 mU/ml GlcNAcT-I

[1442] 100 mU/ml GlcNAcT-II

[1443] 1 mg/ml EPO (purified, expressed in Sf9 cells, purchased fromProtein Sciences).

[1444] Analysis of glycoforms. This assay is a slight modification onK-R Anumula and ST Dhume, Glycobiology 8 (1998) 685-69. N-glycanase(PNGase) released N-glycans were reductively labeled with anthranilicacid. The reductively-aminated N-glycans were injected onto a ShodexAsahipak NH2P-50 4D amino column (4.6 mm×150 mm). Two solvents were usedfor the separation: A) 5% (v/v) acetic acid, 1% tetrahydrofuran, and 3%triethylamine in water, and B) 2% acetic acid and 1% tetrahydrofuran inacetonitrile. The column was then eluted isocratically with 70% B for2.5 minutes, followed by a linear gradient over a period of 97.5 minutesgoing from 70 to 5% B and a final isocratic elution with 5% B for 15minutes. Eluted peaks were detected using fluorescence detection with anexcitation of 230 nm and emission wavelength of 420 nm.

[1445] Under these conditions, the trimannosyl core had a retention timeof 22.3 minutes, and the product of the GnT reaction has a retentiontime of 30 minutes. The starting material was exclusively trimannosylcore with core GlcNAc (FIG. 124).

[1446] 12. Preparation of EPO With Multi-Antennary Complex Glycans

[1447] This example sets forth the preparation of PEGylated, biantennaryEPO, and triantennary, sialylated EPO from insect cell expressed EPO.

[1448] Recombinant human erythropoietin (rhEPO) from the baculovirus/Sf9expression system (Protein Sciences Corp., Meriden, Conn.) was subjectedto glycan analysis and the resulting glycans were shown to be primarilytrimannosyl core with core fucose, with a small percentage of glycansalso having a single GlcNAc.

[1449] Addition of N-acetylglucosamine with GnT-I and GnT-II. Two lotsof rhEPO (1 mg/mL) were incubated with GnT-I and GnT-II, 5 mMUDP-glcNAc, 20 mM MnCl₂, and 0.02% sodium azide in 100 mM MES pH 6.5 at32° C. for 24 hr. Lot A contained 20 mg of EPO, and 100 mU/mL GnT-I and60 mU/mL GnT-II. Lot B contained 41 mg of EPO, and 41 mU/mL GnT-I+50mU/mL GnT-II. After the reaction, the sample was desalted by gelfiltration (PD10 columns, Pharmacia LKB Biotechnology Inc., Piscataway,N.J.).

[1450] EPO glycans analyzed by 2-AA HPLC profiling. This assay is aslight modification on Anumula and Dhume, Glycobiology 8 (1998) 685-69.Reductively-aminated N-glycans were injected onto a Shodex AsahipakNH2P-50 4D amino column (4.6 mm×150 mm). Two solvents were used for theseparation, A) 5% (v/v) acetic acid, 1% tetrahydrofuran, and 3%triethylamine in water and B) 2% acetic acid and 1% tetrahydrofuran inacetonitrile. The column was then eluted isocratically with 70% B for2.5 min, followed by a linear gradient over a period of 100 min goingfrom 70 to 5% B, and a final isocratic elution with 5% B for 20 min.Eluted peaks were detected using fluorescence detection with anexcitation of 230 nm and emission wavelength of 420 nm. Non-sialylatedN-linked glycans fall in the LC range of 23-34 min, monosialylated from34-42 min, disialylated from 42-52 min, trisialylated from 55-65 min andtetrasialylated from 68-78 min.

[1451] Glycan profiling by 2AA HPLC revealed that lot A was 92%converted to a biantennary structure with two GlcNAcs (the balancehaving a single GlcNAc. Lot B showed 97% conversion to the desiredproduct (FIGS. 125A and 125B).

[1452] Introducing a third antennary branch with GnT-V. EPO (1 mg/mL oflot B) from the product of the GnT-I and GnT-II reactions, afterdesalting on PD-10 columns and subsequent concentration, was incubatedwith 10 mU/mL GnT-V and 5 mM UDP-GlcNAc in 100 mM MES pH 6.5 containing5 mM MnCl₂ and 0.02% sodium azide at 32° C. for 24 hrs. 2AA HPLCanalysis demonstrated that the conversion occurred with 92% efficiency(FIG. 126).

[1453] After desalting (PD-10) and concentration, galactose was addedwith rGalTI: EPO (1 mg/mL) was incubated with 0.1 U/mL GaLT1, 5 mMUDP-galactose, 5 mM MnCl₂ at 32° C. for 24 hrs.

[1454] MALDI analysis of reductively-aminated N-glycans from EPO. Asmall aliquot of the PNGase released N-glycans from EPO that had beenreductively labeled with anthranilic acid was dialyzed for 45 min on anMF-Millipore membrane filter (0.025 μm pore, 47 mm dia), which wasfloating on water. The dialyzed aliquot was dried in a speedvac,redissolved in a small amount of water, and mixed with a solution of2,5-dihydroxybenzoic acid (10 g/L) dissolved in water/acetonitrile(50:50). The mixture was dried onto the target and analyzed using anApplied Biosystems DE-Pro MALDI-TOF mass spectrometer operated in thelinear/negative-ion mode. Oligosaccharides were assigned based on theobserved mass-to-charge ratio and literature precedence.

[1455] Analysis of released glycans by MALDI showed that galactose wasadded quantitatively to all available sites (FIG. 127). GalactosylatedEPO from above was then purified by gel filtration on a Superdex 1.6/60column in 50 mM Tris, 0.15M NaCl, pH 6.

[1456] Sialylation. After concentration and desalting (PD-10), 10 mggalactosylated EPO (1 mg/mL) was incubated with ST3Gal3 (0.05 U/mL), andCMP-SA (3 mM) in 50 mM Tris, 150 mM NaCl, pH 7.2 containing 0.02% sodiumazide. A separate aliquot contained radiolabelled CMP-SA. The resultingincorporated label and free label was separated by isocratic sizeexclusion chromatography/HPLC at 0.5 mL/min in 45% MeOH, 0.1% TFA (7.8mm×30 cm column, particle size 5 μm, TSK G2000SWXL, Toso Haas, AnsysTechnologies, Lake Forest, Calif.). Using this procedure, 12% of thecounts were incorporated (360 micromolar, at 33 micromolar EPO, or about10.9 moles/mole). Theoretical (3 N-linked sites, tri-antennary) is about9 moles/mole incorporation. These correspond within the limits of themethod. In an identical reaction with ST6Gal1 instead of ST3Gal3, 5.7%of the radiolabel was incorporated into the galactosylated EPO, or about48% compared with ST3Gal3.

[1457] 13. GlycoPEGylation of EPO Produced in Insect Cells

[1458] This example sets forth the prepartion of PEGylated biantennaryEPO from insect cell expressed EPO.

[1459] Recombinant human erythropoietin (rhEPO) from the baculovirus/Sf9expression system (Protein Sciences Corp., Meriden, Conn.) was subjectedto glycan analysis and the resulting glycans were shown to be primarilytrimannosyl core with core fucose, with a small percentage of glycansalso having a single GlcNAc (FIG. 128).

[1460] Addition of N-acetylglucosamine with GnT-I and GnT-II. Two lotsof rhEPO (1 mg/mL) were incubated with GnT-I and GnT-II, 5 mMUDP-glcNAc, 20 mM MnCl₂, and 0.02% sodium azide in 100 mM MES pH 6.5 at32° C. for 24 hr. Lot A contained 20 mg of EPO, and 100 mU/mL GnT-I and60 mU/mL GnT-II. Lot B contained 41 mg of EPO, and 41 mU/mL GnT-I+50mU/mL GnT-II. After the reaction, the sample was desalted by gelfiltration (PD10 columns, Pharmacia LKB Biotechnology Inc., Piscataway,N.J.).

[1461] Glycan profiling by 2AA HPLC revealed that lot A was 92%converted to a biantennary structure with two GlcNAcs (the balancehaving a single glcNAc. Lot B showed 97% conversion to the desiredproduct (FIGS. 125A and 125B).

[1462] Galactosylation of EPO lot A. EPO (˜16 mgs of lot A) was treatedwith GnT-II to complete the addition of GlcNAc. The reaction was carriedout in 50 mM Tris pH 7.2 containing 150 mM NaCl, EPO mg/ml, 1 mMUDP-GlcNAc, 5 mM MnCl₂, 0.02% sodium azide and 0.02 U/ml GnT-II at 32 Cfor 4 hrs. Then galactosylation of EPO was done by adding UDP-galactoseto 3 mM and GalT1 to 0.5 U/ml and the incubation continued at 32° C. for48 hrs.

[1463] Galactosylated EPO was then purified by gel filtration on aSuperdex75 1.6/60 column in 50 mM Tris, 0.15M NaCl, pH 6. The EPOcontaining peak was then analyzed by 2AA HPLC. Based on the HPLC data˜85% of the glycans contains two galactose and ˜15% of the glycans didnot have any galactose after galactosylation reaction.

[1464] Sialylation of galactosylated EPO. Sialylation of galactosylatedEPO was carried out in 100 mM Tris pH containing 150 mM NaCl, 0.5 mg/mlEPO, 200 mU/ml of ST3Gal3 and either 0.5 mM CMP-SA or CMP-SA-PEG (1 kDa)or CMP-SA-PEG (10 kDa) for 48 hrs at 32° C. Almost all of the glycansthat have two galactose residues were fully sialylated (2 sialicacids/glycan) after sialylation reaction with CMP-SA. MALDI-TOF analysisconfirmed the HPLC data.

[1465] PEGylation of galactosylated EPO. For PEGylation reactions usingCMP-SA-PEG (1 kDa) and CMP-SA-PEG (10 kDa), an aliquot of the reactionmixture was analyzed by SDS-PAGE (FIG. 129). The molecular weight of theEPO peptide increased with the addition of each sugar, and increasedmore dramatically in molecular weight after the PEGylation reactions.

[1466] In vitro bioassay of EPO. In vitro EPO bioassay (adapted fromHammerling et al, 1996, J. Pharm. Biomed. Anal. 14: 1455-1469) is basedon the responsiveness of the TF-1 cell line to multiple levels of EPO.TF-1 cells provide a good system for investigating the proliferation anddifferentiation of myeloid progenitor cells. This cell line wasestablished by T. Kitamura et al. in October 1987 from a heparinizedbone marrow aspiration sample from a 35 year old Japanese male withsevere pancytopenia. These cells are completely dependent on Interleukin3 or Granulocyte-macrophage colony-stimulating factor (GM-CSF).

[1467] The TF-1 cell line (ATCC, Cat. No. CRL-2003) was grown inRPMI+FBS 10%+GM-CSF (12 ng/ml) and incubated at 37° C. 5% CO₂. The cellswere in suspension at a concentration of 5000 cells/ml of media, and 200μl were dispensed in a 96 well plate. The cells were incubated withvarious concentrations of EPO (0.1 μg/ml to 10 μg/ml) for 48 hours. AMTT Viability Assay was then done by adding 25 μl of MTT at 5 mg/ml(SIGMA M5655), incubating the plate at 37° C. for 20 min to 4 hours,adding 100 μl of isopropanol/HCl solution (100 ml isopropanol+333 μl HCl6N), reading the OD at 570 nm, and 630 nm or 690 nm, and subtracting thereadings at 630 nm or 690 nm from the readings at 570 nm.

[1468]FIG. 130 contains the results when sialylated EPO, and EPOglycoPEGylated with 1 kDa or 10 kDa PEG was subjected to an in vitro EPObioactivity test. The EPO glycoPEGylated with 1 kDa PEG had almost thesame activity as the unglycoPEGylated EPO when both were at aconcentration of approximately 5 μg/ml. The EPO glycoPEGylated with 10kDa PEG had approximately half the activity of the unglycoPEGylated EPOwhen both were at a concentration of approximately 5 μg/ml.

[1469] 14. GlycoPEGylation of O-Linked Glycans of EPO Produced in CHOCells

[1470] Preparation of O-linked EPO-SA-PEG (10 kDa). Asialo-EPO,originally produced in CHO cells, is dissolved at 2.5 mg/mL in 50 mMTris-HCl, 0.15 M NaCl, 0.05% NaN₃, pH 7.2. The solution is incubatedwith 5 mM CMP-SA and 0.1 U/mL of ST3Gal3 at 32° C. for 2 days. Tomonitor the incorporation of sialic acid onto the N-linked glycans, asmall aliquot of the reaction had CMP-SA-14C added; the peptide isseparated by gel filtration on a Toso Haas G2000SW analytical columnusing methanol, water and the product detected using a radiationdetector. When the reaction is complete, the solution is concentratedusing a Centricon-20 filter. The remaining solution is buffer exchangedwith 0.05 M Tris (pH 7.2), 0.15 M NaCl, 0.05% NaN₃ to a final volume of7.2 mL until the CMP-SA could no longer be detected. The retentate isthen resuspended in 0.05 M Tris (pH 7.2), 0.15 M NaCl, 0.05% NaN₃ at 2.5mg/mL protein. The solution is incubated with 1 mM CMP-SA-PEG (10 kDa)and ST3Gal1, to glycosylate the O-linked site, at 32° C. for 2 days. Tomonitor the incorporation of sialic acid-PEG, a small aliquot of thereaction is separated by gel filtration suing a Toso Haas TSK-gel-3000analytical column eluting with PBS pH 7.0 and analyzing by UV detection.When the reaction is complete, the reaction mixture is purified using aToso Haas TSK-gel-3000 preparative column using PBS buffer (pH 7.0)collecting fractions based on UV absorption. The product of the reactionis analyzed using SDS-PAGE and IEF analysis according to the proceduresand reagents supplied by Invitrogen. Samples are dialyzed against waterand analyzed by MALDI-TOF MS.

[1471] 15. EPO-Transferrin

[1472] This example sets forth the procedures for the glycoconjugationof proteins to O-linked glycans, and in particular, transferrin isglycoconjugated to EPO. The sialic acid residue is removed from O-linkedglycan of EPO, and EPO-SA-linker-SA-CMP is prepared.EPO-SA-linker-SA-CMP is glycoconjugated to asialotransferrin withST3Gal3.

[1473] Preparation of O-linked asialo-EPO. EPO (erythropoietin) producedin CHO cells is dissolved at 2.5 mg/mL in 50 mM Tris 50 mM Tris-HCl pH7.4, 0.15 M NaCl, and is incubated with 300 mU/mL sialidase (Vibriocholera)-agarose conjugate for 16 hours at 32° C. To monitor thereaction a small aliquot of the reaction is diluted with the appropriatebuffer and a IEF gel performed according to Invitrogen procedures. Themixture is centrifuged at 10,000 rpm and the supernatant is collected.The supernatant is concentrated to a EPO concentration of about 2.5mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN₃, pH 7.2. The solutionis incubated with 5 mM CMP-sialic acid and 0.1 U/mL of ST3Gal3 at 32° C.for 2 days. To monitor the incorporation of sialic acid, a small aliquotof the reaction had CMP-SA-fluorescent ligand added; the labelincorporated into the peptide is separated from the free label by gelfiltration on a Toso Haas G3000SW analytical column using PBS buffer (pH7.1). When the reaction is complete, the reaction mixture is purifiedusing a Toso Haas G3000SW preparative column using PBS buffer (pH 7.1)and collecting fractions based on UV absorption. The product of thereaction is analyzed using SDS-PAGE and IEF analysis according to theprocedures and reagents supplied by Invitrogen. Samples are dialyzedagainst water and analyzed by MALDI-TOF MS.

[1474] Preparation of EPO-SA-linker-SA-CMP. The O-linked asialo-EPO 2.5mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN₃, pH 7.2. The solutionis incubated with 1 mM CMP-sialic acid-linker-SA-CMP and 0.1 U/mL ofST3Gal1 at 32° C. for 2 days. To monitor the incorporation of sialicacid-linker-SA-CMP, the peptide is separated by gel filtration on a TosoHaas G3000SW analytical column using PBS buffer (pH 7.1).

[1475] After 2 days, the reaction mixture is purified using a Toso HaasG3000SW preparative column using PBS buffer (pH 7.1) and collectingfractions based on UV absorption. The product of the reaction isanalyzed using SDS-PAGE and IEF analysis according to the procedures andreagents supplied by Invitrogen. Samples are dialyzed against water andanalyzed by MALDI-TOF MS.

[1476] Preparation of Transferrin-SA-Linker-SA-EPO. EPO-SA-Linker-SA-CMPfrom above is dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl,0.05% NaN₃, pH 7.2. The solution is incubated with 2.5 mg/mLasialo-transferrin and 0.1 U/mL of ST3Gal3 at 32° C. for 2 days. Tomonitor the incorporation of transferrin, the peptide is separated bygel filtration on a Toso Haas G3000SW analytical column using PBS buffer(pH 7.1) and the product detected by UV absorption. When the reaction iscomplete, the solution is incubated with 5 mM CMP-SA and 0.1 U/mL ofST3Gal3 (to cap any unreacted transferrin glycans) at 32° C. for 2 days.The reaction mixture is purified using a Toso Haas G3000SW preparativecolumn using PBS buffer (pH 7.1) collecting fractions based on UVabsorption. The product of the reaction is analyzed using SDS-PAGE andIEF analysis according to the procedures and reagents supplied byInvitrogen. Samples are dialyzed against water and analyzed by MALDI-TOFMS.

[1477] 16. EPO-GDNF

[1478] This example sets forth the procedures for the glycoconjugationof proteins, and in particular, the preparation ofEPO-SA-Linker-SA-GDNF.

[1479] Preparation of EPO-SA-Linker-SA-GDNF. EPO-SA-Linker-SA-CMP fromabove is dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05%NaN₃, pH 7.2. The solution is incubated with 2.5 mg/mL GDNF (produced inNSO) and 0.1 U/mL of ST3Gal3 at 32° C. for 2 days. To monitor theincorporation of GDNF, the peptide is separated by gel filtration on aToso Haas G3000SW analytical column using PBS buffer (pH 7.1) and theproduct detected by UV absorption. When the reaction is complete, thesolution is incubated with 5 mM CMP-SA and 0.1 U/mL of ST3Gal3 (to capany unreacted GDNF glycans) at 32° C. for 2 days. The reaction mixtureis purified using a Toso Haas G3000SW preparative column using PBSbuffer (pH 7.1) collecting fractions based on UV absorption. The productof the reaction is analyzed using SDS-PAGE and IEF analysis according tothe procedures and reagents supplied by Invitrogen. Samples are dialyzedagainst water and analyzed by MALDI-TOF MS.

[1480] 17. Mono-Antennary GlycoPEGylation of EPO

[1481] This example sets forth the procedure for the preparation ofglycoPEGylated mono-antennary erythropoietin (EPO), and its bioactivityin vitro and in vivo.

[1482] When EPO (GenBank Accession No. P01588) is expressed in CHOcells, N-linked glycans are formed at amino acid residues 24, 38 and 83,and an O-linked glycan is formed at amino acid residue 126 (FIG. 131;Lai et al., 1986, J. Biol. Chem. 261:3116-3121). The bioactivity of thisglycoprotein is directly correlated with the level of NeuAc content.Increased sialic acid decreases the binding of EPO to its receptor invitro; however increased sialic acid increases the bioactivity of EPO invivo. The O-linked glycan has no impact on the in vitro or in vivoactivity of EPO, or the pharmacokinetics of the molecule (Wasley et al.,1991, Blood 77:2624-2632).

[1483] When EPO is expressed in insect cells, such as is accomplishedusing a baculovirus/Sf9 expression system (see also, Wojchowshi et al.,1987, Biochem. Biophys. Acta 910:224-232; Quelle et al., 1989, Blood74:652-657), N-linked glycans are formed at amino acid residues 24, 38and 83, but an O-linked glycan is not formed at amino acid residue 126(FIG. 132). This is because the insect cell does not have a glycosyltransferase that recognizes the amino acid sequence around amino acidresidue 126 of EPO. The majority of the N-linked glycans are composed ofGlcNAc₂Man₃Fuc. In the present example, EPO expressed in insect cellswas remodeled with high efficiency to achieve the complex glycanSA₂Gal₂GlcNAc₂Man₃FucGlcNAc₂ by contacting the protein with, in series,GnT1,2, GalT-1, and ST in the presence of the appropriate donormolecules. These enzymatic reactions were performed on insect cellexpressed EPO using reaction conditions disclosed herein, to yield thecomplex glycans herein with 92% total efficiency (Table 21). Optionally,O-linked glycans can also be added (O'Connell and Tabak, 1993, J. Dent.Res. 72:1554-1558; Wang et al., 1993, J. Biol. Chem. 268:22979-22983).TABLE 21 Percent of each species of glycan structure in the populationof glycan structures on insect cell expressed EPO (“starting material”),and on EPO after each sequential enzymatic remodeling step. StartingAfter After Glycan Material GnT-I, II GalT-I After ST

0.5%

98.0%

1.0% 0.5%  0.5%

0.5% 99.5%   4% 2%

95.9% 5%

92.0%  

= fucose,

= GlcNAc,

= mannose,

= galactose,

= N-acetylneuraminic acid

[1484] Also in the present example, EPO expressed in insect cells wasremodeled to form mono-antennary, bi-anntenary and tri-antennaryglycans, which were subsequently glycoPEGylated with 1 kDa, 10 kDa and20 kDa PEG molecules suing procedures described elsewhere herein. Themolecular weights of these EPO forms were determined, and were comparedto Epoetin™ having 3 N-linked glycans, and NESP (Aranesp™) having 5N-linked glycans (FIG. 133). Examples of the preparation of bi- andtri-antennary glycan structures are given in Example 7, herein.

[1485] EPO having monoantennary PEGylated glycan structures is preparedby expressing EPO peptide in insect cells, then contacting the EPOpeptide with GnTI only (or alternatively GnTII only) in the presence ofa GlcNAc donor. The EPO peptide is then contacted with GalT-I in thepresence of a galactose donor. The EPO peptide is then contacted with STin the presence of SA-PEG donor molecules (FIG. 134A) to generate an EPOpeptide having three N-linked mono-antennary PEGylated glycan structures(FIG. 134B).

[1486] The in vitro bioactivity of EPO-SA and EPO-SA-PEG generated frominsect cell expressed EPO was accessed by measuring the ability of themolecule to stimulate the proliferation of TF-1 erythroleukemia cells.Tri-antennary EPO-SA-PEG 1 kDa exhibited almost all of the bioactivityof tri-antennary EPO-SA, and di-antennary EPO-SA-PEG 10 kDa exhibitedalmost all of the bioactivity of di-antennary EPO-SA over a range of EPOconcentrations (FIG. 135). Remodeled and glycoPEGylated EPO generated ininsect cells exhibited up to 94% of the in vitro bioactivity of Epogen™,which is EPO expressed in CHO cells without further glycan remodeling orPEGylation (Table 22). TABLE 22 In vitro activity of the EPO constructsas compared with Epogen ™ at 2 μg/ml protein and 48 hr. Compound (2μg/ml protein) Activity (percent of Epogen ™) Biantennary-SA 146Biantennary-SA-PEG 1K 94 Biantennary-SA-PEG 10K 75 Triantennary-SA 2,3¹42 Triantennary-SA-PEG 1K 48 Triantennary-SA-PEG 10K 34

[1487] The in vivo pharmacokinetics of glycoPEGylated andnon-glycoPEGylated EPO was determined. GlycoPEGylated andnon-glycoPEGylated [I¹²⁵]-labeled EPO was bolus injected into rats andthe pharmacokinetics of the molecules were determined. As compared withbi-antennary EPO, the AUC of bi-antennary EPO-PEG 1 kDa was 1.8 timesgreater, and the AUC of bi-antennary EPO-PEG 10 kDa was 11 times greater(FIG. 136). As compared with bi-antennary EPO, the AUC of bi-antennaryEPO-PEG 1 kDa was 1.6 times greater, and the AUC of bi-antennary EPO-PEG10 kDa was 46 times greater (FIG. 136). Therefore, the pharmacokineticsof EPO was greatly improved by glycoPEGylation.

[1488] The in vivo bioactivity of glycoPEGylated and non-glycoPEGylatedEPO was also determined by measuring the degree to which the EPOconstruct could stimulate reticulocytosis. Reticulocytosis is a measureof the rate of the maturation of red blood cell precursor cells intomature red blood cells (erythrocyte). Eight mice per treatment groupwere given a single subcutaneous injection of 10 μg protein/Kg, and thepercent reticulocytes was measured at 96 hours (FIG. 137). Tri- andbi-antennary PEGylated EPO exhibited greater in vivo bioactivity thannon-PEGylated EPO forms, including Epogen™.

[1489] Further determination of in vivo bioactivity of the EPOconstructs was assessed by measuring the hematocrit (the percent ofwhole blood that is comprised of red blood cells) of CD-I female mice 15days after intraperitoneal injection three times per week with 2.5 μgpeptide/kg body weight of the EPO construct. The hematocrit incrementincreased with the size of the EPO form, with the 82.7 kDamono-antennary EPO-PEG 20 kDa having a slightly greater activity thanthe 35.6 kDa NESP (Aranesp™) and about two times the bioactivity of 28.5kDa Epogen™ (FIG. 138).

[1490] This example illustrates that the generation of a longer-actingglycoPEGylated EPO is feasible. The pharmacokinetic profile ofglycoPEGylated EPO can be customized by altering the number ofglycoPEGylation sites and the size of the PEG molecule added to alterthe half-life of the peptide in the bloodstream. Finally, glycoPEGylatedEPO retains both in vitro and in vivo bioactivity.

[1491] 18. Preparation and Bioactivity of Sialylated and PEGylatedMono-, Bi- and Tri-Antennary EPO

[1492] This example illustrates the production of glycoPEGylated EPO, inparticular PEGylated EPO having mono-antennary and bi-antennary glycanswith PEG linked thereto. The following EPO variants were produced:mono-antennary PEG (1 kDa) and PEG (20 kDa); bi-antennary 2,3-sialicacid (SA), bi-antennary SA-PEG (1 kDa), bi-antennary SA-PEG (10 kDa);tri-antennary 2,3-SA and tri-antennary 2,6-SA capped with 2,3-SA.

[1493] Recombinant erythropoietin (rEPO) expressed in insect cells wasobtained from Protein Sciences (Lot # 060302, Meridan Conn.). The glycancomposition of this batch of EPO had approximately 98% trimannosyl corestructure. FIG. 139A depicts the HPLC analysis of the released glycansfrom this EPO, with peak “P2” representing the trimannosyl core glycan.FIG. 139B shows the MALDI analysis of the released glycans with thestructures of the released glycans beside the peak they represent.

[1494] Mono-Antennary Branching

[1495] Several steps were performed to produce the mono-antennarybranched structure. In brief, the first step was a GnT-I/GalT-1 reactionfollowed by purification using Superdex-75 chromatography. This reactionadds a GlcNAc moiety to one branch of the tri-mannosyl core, and agalactose moiety onto the GlcNAc moiety. Branching was extended with theST3Gal3 reaction to add the SA-PEG (10 kDa) moiety or the SA-PEG (20kDa) moiety onto the terminal galactose moiety. The final purificationwas accomplished using Superdex-200 chromatography (AmershamBiosciences, Arlington Heights, Ill.).

[1496] GnT-I/GalT-1 Reaction. The GnT-I and GalT-1 reactions werecombined and incubated at 32° C. for 36 hours. The reaction contained 1mg/nL EPO, 100 mM Tris-Cl pH 7.2, 150 mM NaCl, 5 mM MnCl₂, 0.02% NaN₃, 3mM UDP-GlcNAc, 50 mU/mg GnT-1,3 mM UDP-Gal, and 200 mU/mg GalT-1. FIG.140 depicts the MALDI analysis of glycans released from EPO after theGnT-I/GalT-1 reaction. Glycan analysis showed approximately 90% of theglycans had the desired mono-antennary branched structure with aterminal galactose moiety.

[1497] Superdex 75 Purification. After the GnT-I/GalT1 reaction, EPO waspurified from the enzyme protein contaminants and nucleotide sugarsusing a 1.6 cm×60 cm Superdex-75 gel filtration chromatography (AmershamBiosciences, Arlington Heights, Ill.) in PBS containing 0.02% Tween 20(Sigma-Aldrich Corp., St. Louis, Mo.).

[1498] ST3Gal3 Reaction. The ST3Gal3 PEGylation reaction was incubatedat 32° C. for 24 hours. The reaction contained 1 mg/mL EPO, 100 mMTris-Cl pH 7.2, 150 mM NaCl, 0.02% NaN₃, 200 mU/mg ST3Gal3, and 0.5 mMCMP-SA-PEG (10 kDa) or 0.5 mM CMP-SA-PEG (20 kDa). FIG. 141 depicts theSDS-PAGE analysis of EPO after this reaction. The correspondingmolecular weights of the protein bands indicate that the EPO glycansformed by the GnT-I/GalT-1 reaction were completely sialylated with thePEG derivative.

[1499] Superdex 200 Purification. EPO then was purified from thecontaminants of the ST3Gal3 reaction by a 1.6 cm×60 cm Superdex-200 gelfiltration chromatography (Amersham Biosciences, Arlington Heights,Ill.) in PBS containing 0.02% Tween-20.

[1500] TF-1 Cell In Vitro Bioassay of Mono-antennary PEGylated EPO. TheTF-1 cell line is used to assess the activity of EPO in vitro. The TF-1cells line is a myeloid progenitor cell line available from the AmericanType Culture Collection (Catalogue No. CRL-2003, Rockville, Md.). Thecell line is completely dependant on Interleukin-3 orGranulocyte-Macrophage Colony-Stimulating Factor for viability. TF-1cells provide a good system for investigating the effect of EPO onproliferation and differentiation.

[1501] The TF-1 cells were grown in RPMI with 10% FBS and 12 ng/mlGM-CSF at 37° C. in 5% CO₂. The cells were suspended at a concentrationof 10,000 cells/ml of media. 200 μl aliquots of cells were dispensedinto a 96-well plate. The cells were incubated with 0.1 to 10 μg/ml EPOfor 48 hrs.

[1502] The MTT viability assay was then performed by first adding 25 μlof 5 μg/ml MTT (3-[4,5-dimethlythiazol-2-yl]-2,5-diphenyltetrazoliumbromide, or thiazolyl blue; Sigma Chemical Co., St. Louis, Mo.,Catalogue No. M5655). The plate was incubated for 4 hrs at 37° C. 100 μlof isopropanol/HCl solution (100 ml isopropanol and 333 μl HCl 6N) wasadded. The absorbency of the plates was read at 570 nm and either 630 or690 nm, and the reading at either 630 nm or 690 nm was subtracted forthe reading at 570 nm.

[1503]FIG. 142 depicts the results of the bioassay of EPO activity afterPEGylation of it mono-antennary glycans. In this bioassay, themono-antennary PEGylated EPO is much less active that a non-PEGylatedEPO (Epogen).

[1504] Bi-Antennary Branching

[1505] Several reactions were performed to accomplish the bi-antennarybranching of EPO. Briefly, the first reaction combined the GnT-I andGnT-II reactions to add GlcNAc moieties to two of the tri-mannosyl corebranches. The second reaction, the GalT-1 reaction, adds a galactosemoiety to each GlcNAc moieties. Superdex 75 chromatography (AmershamBiosciences, Arlington Heights, Ill.) was performed prior to the ST3Gal3reaction. The bi-antennary branching was further extended with theST3Gal3 reaction to add either a 2,3-SA, or SA-PEG (1 kDa), SA-PEG (10kDa). Final purification was accomplished using Superdex 200chromatagraphy (Amersham Biosciences, Arlington Heights, Ill.).

[1506] GnT-I/GnT-II Reaction. The GnT-I and GnT-II reactions werecombined and incubated at 32° C. for 48 hours. The reaction contained 1mg/mL EPO, 100 mM MES pH 6.5, 150 mM NaCl, 20 mM MnCl₂, 0.02% NaN₃, 5 mMUDP-GlcNAc, 100 mU/mg GnT-I, 60 mU/mg GnT-II. The reaction achieved 92%completion of the addition of bi-antennary GlcNAc moieties, with 8%mono-antennary GlcNAc moieties. FIG. 143A shows the HPLC analysis of thereleased glycans, where peak “P3” represents the bi-antennary GlcNAcglycan. FIG. 143B depicts the MALDI analysis of the released glycanswith the structures of the glycans indicated beside the peak that theyrepresent.

[1507] In order to further the reaction, an additional 20 mU/mg ofGnT-II was added along with 1 mM UDP-GlcNAc, 5 mM MnCl₂, 0.02% NaN₃, andthe mixure was incubated for 4 hours at 32° C. Greater than 99% of thisreaction achieved completion of the bi-antennary GlcNAc glycan.

[1508] GalT-1 Reaction. The GalT-1 reaction was started immediatelyafter the completion of the second GnT-II reaction. Enzyme andnucleotide sugar were added to the completed GnT-II reaction atconcentrations of 0.5 U/mg GalT-1 and 3 mM UDP-Gal.

[1509] When the GalT-1 reaction was performed on a small scale, withabout 100 μg EPO per reaction, approximately 95% of the reactionproduced EPO with bi-antennary terminal galactose moiety. FIG. 144Adepicts the HPLC analysis of the released glycans where peak “P2” is thebi-antennary glycan with terminal galactose moieties (85% of theglycans), and peak “P1” is the bi-antennary glycan without the terminalgalactose moieties (15% of the glycans).

[1510] The GalT-1 reaction was also performed on a large scale withabout 16 mg of EPO per reaction. FIG. 144B depicts the HPLC analysis ofthe release glycans from the large scale GalT-1 reaction, where peak“P2” is the bi-antennary glycan with terminal galactose moieties, andpeak “P1” is the bi-antennary glycan without the terminal galactosemoieties.

[1511] Superdex 75 Purification. EPO was then purified from the enzymeprotein contaminants and nucleotide sugars using a 1.6 cm×60 cmSuperdex-75 gel filtration chromatography (Amersham Biosciences,Arlington Heights, Ill.) in PBS containing 0.02% Tween 20 after theGnT-II/GalT1 reaction. FIG. 145 depicts the chromatogram of the Superdex75 gel filtration, where peak 2 is EPO with bi-antennary glycans withterminal galactose moieties. FIG. 146 shows SDS-PAGE analysis of theproducts of each remodeling step indicating the increase in themolecular weight of EPO with each remodeling step.

[1512] ST3Gal3 Reaction. The ST3Gal3 reaction was incubated at 32° C.for 24 hours. The reaction contained 0.5 mg/mL EPO, 100 mM Tris-Cl pH7.2, 150 mM NaCl, 0.02% NaN₃, 100 mU/mg ST3Gal3, and 0.5 mM CMP-SA, 0.5mM CMP-SA-PEG (1 kDa), or 0.5 mM CMP-SA-PEG (10 kDa). FIG. 147 shows theresults of SDS-PAGE analysis of EPO before and after the ST3Gal3reaction. Based on this SDS-PAGE analysis, bi-antennary EPO containingterminal Gal can no longer be visually detected after each ST3Gal3reaction. All sialylated EPO variants show an increase in size comparedto non-sialylated EPO at the start of the reaction.

[1513] Superdex 200 Purification. EPO was purified from the contaminantsof the ST3Gal3 reactions by a 1.6 cm×60 cm Superdex-200 gel filtrationchromatography (Amersham Biosciences, Arlington Heights, Ill.) in PBScontaining 0.02% Tween-20. Table 23 summaries the distribution of glycanstructures at each remodeling step. TABLE 23 Summary of glycanstructures on EPO after each remodeling step. Starting After GnT-I After2nd After Glycan Material and GnT-II GnT-II GalT-1 After ST

0.5%

98.0%

1.0% 8.0% 0.5% 0.5% 0.5%

0.5% 92.0% 99.5% 15.5% 15.5%

84.0% 2.0%

82.0%

[1514] Diamonds represent fucose, and squares represent GlcNAc, circlesrepresent mannose, open circles represent galactose.

[1515] Tri-Antennary Branching

[1516] Several reactions were performed to accomplish the tri-antennarybranching of EPO. Briefly, the first reaction combined the GnT-I andGnT-II reactions to add a GlcNAc moiety to the two outer tri-mannosylcore branches of the glycan. The second reaction, GnT-V reaction, adds asecond GlcNAc moiety to one of the two outer trimannosyl core branchesso that there are now three GlcNAc moieties. The third reaction, GalT-1reaction, adds a galactose moiety to each terminal GlcNAc moiety. TheEPO products were then separated by Superdex 75 chromatography. Thetri-antennary branching was further extended with the ST3Gal3 reactionto add either a 2,3-SA moiety or a 2,6-SA moiety, and capped with a2,3-SA moiety. Final purification was accomplished using Superdex 75chromatography.

[1517] GnT-I/GnT-II Reaction. The GnT-I and GnT-II reactions werecombined and incubated at 32° C. for 24 hours. The reaction contained 1mg/mL EPO, 100 mM MES pH 6.5, 150 mM NaCl, 20 mM MnCl₂, 0.02% NaN₃, 5 mMUDP GlcNAc, 50 mU/mg GnT-I and 41 mU/mg GnT-II. The reaction achieved97% completion of the addition of the bi-antennary GlcNAc moiety, with3% tri-mannosyl core remaining. FIG. 148 depicts the HPLC analysis ofthe glycans released from EPO after the GnT-I/GnT-II reaction.

[1518] GnT-V Reaction. The GnT-V reaction containing 100 mM MES pH 6.5,5 mM UDP-GlcNAc, 5 mM MnCl₂, 0.02% NaN₃, 10 mU/mg GnT-V and 1 mg/mL EPO,was incubated at 32° C. for 24 hours. This reaction adds a GlcNAc moietyto an outer mannose moiety already containing a GlcNAc moiety. FIG. 149depicts the HPLC analysis of the glycans released from EPO after theGnT-V reaction. Approximately 92% the glycans released from EPO were thedesired product, tri-antennary branched EPO with terminal GlcNAcmoieties, based on glycan and MALDI analysis. The remaining 8% of theglycans were bi-antennary branched structures containing terminal GlcNAcmoieties.

[1519] GalT-1 Reaction. The GalT-I reaction containing 100 mM Tris pH7.2, 150 mM NaCl, 5 mM UDP Gal, 100 mU/mg GalT-1,5 mM MnCl₂, 0.02% NaN₃and 1 mg/mL EPO was incubated at 32° C. for 24 hours. FIG. 150 depictsthe HPLC analysis of the glycans released from EPO after this reaction.Glycan and MALDI analysis indicates that 97% of the released glycans hadterminal galactose moieties on the tri-antennary branched structures.The remaining 3% was a bi-antennary structure containing a terminalgalactose.

[1520] Superdex 75 Purification. After the GnT-I/GalT1 reaction, EPO waspurified from the enzyme protein contaminants and nucleotide sugarsusing a 1.6 cm×60 cm Superdex-75 gel filtration chromatography (AmershamBiosciences, Arlington Heights, Ill.) in PBS containing 0.02% Tween 20.The purified material was divided into two batches to produce thetri-antennary glycan with terminal 2,6-SA moieties and the tri-antennaryglycan with terminal 2,6-SA moieties capped with 2,6-SA moieties.

[1521] ST3Gal3 Reaction. The ST3Gal3 reaction was incubated at 32° C.for 24 hours. The reaction contained 1 mg/mL galactosylated EPO, 100 mMTris-Cl pH 7.2, 150 mM NaCl, 0.02% NaN₃, 50 mU/mg ST3Gal3, and 3 mMCMP-SA. FIG. 151 depicts the HPLC analysis of glycans released from EPOafter this step. Based on glycan and MALDI analysis, approximately 80%of the released glycans were tri-antennary branched structures withterminal 2,3-SA moieties. The remaining 20% of the released glycans werebi-antennary structures with terminal 2,3-SA moieties.

[1522] ST6Gal1 sialylation Reaction following the ST3Gal3 Reaction. TheST6Gal1 reaction was incubated at 32° C. for 24 hours. The reactioncontained 1 mg/mL sialylated galactosylated EPO, 100 mM Tris-Cl pH 7.2,150 mM NaCl, 0.02% NaN₃, 50 mU/mg ST6Gal1, and 3 mM CMP-SA. FIG. 152depicts the results of HPLC analysis of the glycans released from EPOafter the ST6Gal1 reaction. Based on glycan and MALDI analysis,approximately 80% of the tri-antennary branched glycans containedterminal 2,3-SA moieties. The remaining 20% of the glycans werebi-antennary with terminal 2,3-SA moieties.

[1523] Superdex 75 Purification. EPO was purified from the contaminantsof the ST3Gal3 reactions by a 1.6 cm×60 cm Superdex-75 gel filtrationchromatography (Amersham Biosciences, Arlington Heights, Ill.) in PBScontaining 0.02% Tween-20.

[1524] Bioassay of Tri-antennary and Bi-antennary Sialylated orPEGylated EPO. The activity of the tri-antennary and bi-antennarysialylated EPO glycoforms, and the PEG 10 kDa and 1 kDa bi-antennaryglycoforms were assayed using the TF-1 cell line and the MTT viabilitytest, as described above. FIG. 153 depicts the results of the MTT cellproliferation assay. At 2 μg/ml EDP, the bi-antennary sialylated EPO hadnearly the activity of the control Epogen, while the tri-antennarysialylated EPO had significanly less activity.

[1525] Factor IX

[1526] 19. GlycoPEGylation of Factor IX Produced in CHO Cells

[1527] This example sets forth the preparation of asialoFactor IX andits sialylation with CMP-sialic acid-PEG.

[1528] Desialylation of rFactor IX. A recombinant form of CoagulationFactor IX (rFactor IX) was made in CHO cells. 6000 IU of rFactor IX weredissolved in a total of 12 mL USP H₂O. This solution was transferred toa Centricon Plus 20, PL-10 centrifugal filter with another 6 mL USP H₂O.The solution was concentrated to 2 mL and then diluted with 15 mL 50 mMTris-HCl pH 7.4, 0.15 M NaCl, 5 mM CaCl₂, 0.05% NaN₃ and thenreconcentrated. The dilution/concentration was repeated 4 times toeffectively change the buffer to a final volume of 3.0 mL. Of thissolution, 2.9 mL (about 29 mg of rFactor IX) was transferred to a smallplastic tube and to it was added 530 mU α2-3,6,8-Neuraminidase-agaroseconjugate (Vibrio cholerae, Calbiochem, 450 μL). The reaction mixturewas rotated gently for 26.5 hours at 32° C. The mixture was centrifuged2 minutes at 10,000 rpm and the supernatant was collected. The agarosebeads (containing neuraminidase) were washed 6 times with 0.5 mL 50 mMTris-HCl pH 7.12, 1 M NaCl, 0.05% NaN₃. The pooled washings andsupernatants were centrifuged again for 2 minutes at 10,000 rpm toremove any residual agarose resin. The pooled, desialylated proteinsolution was diluted to 19 mL with the same buffer and concentrated downto ˜2 mL in a Centricon Plus 20 PL-10 centrifugal filter. The solutionwas twice diluted with 15 mL of 50 mM Tris-HCl pH 7.4, 0.15 M NaCl,0.05% NaN₃ and reconcentrated to 2 mL. The final desialyated rFactor IXsolution was diluted to 3 mL final volume (˜10 mg/mL) with the TrisBuffer. Native and desialylated rFactor IX samples were analyzed byIEF-Electrophoresis. Isoelectric Focusing Gels (pH 3-7) were run using1.5 μL (15 μg) samples first diluted with 10 μL Tris buffer and mixedwith 12 μL sample loading buffer. Gels were loaded, run and fixed usingstandard procedures. Gels were stained with Colloidal Blue Stain (FIG.154), showing a band for desialylated Factor IX.

[1529] Preparation of PEG (1 kDa and 10 kDa)-SA-Factor IX. DesialylatedrFactor-IX (29 mg, 3 mL) was divided into two 1.5 mL (14.5 mg) samplesin two 15 mL centrifuge tubes. Each solution was diluted with 12.67 mL50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 0.05% NaN₃ and either CMP-SA-PEG-1kor 10k (7.25 μmol) was added. The tubes were inverted gently to mix and2.9 U ST3Gal3 (326 μL) was added (total volume 14.5 mL). The tubes wereinverted again and rotated gently for 65 hours at 32° C. The reactionswere stopped by freezing at −20° C. 10 μg samples of the reactions wereanalyzed by SDS-PAGE. The PEGylated proteins were purified on a TosoHaas Biosep G3000SW (21.5×30 cm, 13 um) HPLC column with Dulbecco'sPhosphate Buffered Saline, pH 7.1 (Gibco), 6 mL/min. The reaction andpurification were monitored using SDS Page and IEF gels. NovexTris-Glycine 4-20% 1 mm gels were loaded with 10 μL (10 μg) of samplesafter dilution with 2 μL of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05%NaN₃ buffer and mixing with 12 μL sample loading buffer and 1 μL 0.5 MDTT and heated for 6 minutes at 85° C. Gels were stained with ColloidalBlue Stain (FIG. 155) showing a band for PEG (1 kDa and 10kDa)—SA-Factor IX.

[1530] 20. Direct Sialyl-GlycoPEGylation of Factor IX

[1531] This example sets forth the preparation of sialyl-PEGylation ofFactor IX without prior sialidase treatment.

[1532] Sialyl-PEGylation of Factor-IX with CMP-SA-PEG-(10 KDa). FactorIX (1100 IU), which was expressed in CHO cells and was fully sialylated,was dissolved in 5 mL of 20 mM histidine, 520 mM glycine, 2% sucrose,0.05% NaN₃ and 0.01% polysorbate 80, pH 5.0. The CMP-SA-PEG-(10 kDa) (27mg, 2.5 μmol) was then dissolved in the solution and 1 U of ST3Gal3 wasadded. The reaction was complete after gently mixing for 28 hours at 32°C. The reaction was analyzed by SDS-PAGE as described by Invitrogen. Theproduct protein was purified on an Amersham Superdex 200 (10×300 mm, 13μm) HPLC column with phosphate buffered saline, pH 7.0 (PBS), 1 mL/min.R_(t)=9.5 mm.

[1533] Sialyl-PEGylation of Factor-IX with CMP-SA-PEG-(20 kDa). FactorIX (1100 IU), which was expressed in CHO cells and was fully sialylated,was dissolved in 5 mL of 20 mM histidine, 520 mM glycine, 2% sucrose,0.05% NaN₃ and 0.01% polysorbate 80, pH 5.0. The CMP-SA-PEG-(20 kDa) (50mg, 2.3 μmol) was then dissolved in the solution and CST-II was added.The reaction mixture was complete after gently mixing for 42 hours at32° C. The reaction was analyzed by SDS-PAGE as described by Invitrogen.

[1534] The product protein was purified on an Amersham Superdex 200(10×300 mm, 13 μm) HPLC column with phosphate buffered saline, pH 7.0(Fisher), 1 mL/min. R_(t)=8.6 min.

[1535] 21. Sialic Acid Capping of GlycoPEGylated Factor IX

[1536] This examples sets forth the procedure for sialic acid capping ofsialyl-glycoPEGylated peptides. Here, Factor-IX is the exemplarypeptide.

[1537] Sialic acid capping of N-linked and O-linked Glycans ofFactor-IX-SA-PEG (10 kDa). Purified r-Factor-IX-PEG (10 kDa) (2.4 mg)was concentrated in a Centricon® Plus 20 PL-10 (Millipore Corp.,Bedford, Mass.) centrifugal filter and the buffer was changed to 50 mMTris-HCl pH 7.2, 0.15 M NaCl, 0.05% NaN₃ to a final volume of 1.85 mL.The protein solution was diluted with 372 μL of the same Tris buffer and7.4 mg CMP-SA (12 μmol) was added as a solid. The solution was invertedgently to mix and 0.1 U ST3Gal 1 and 0.1 U ST3Gal3 were added. Thereaction mixture was rotated gently for 42 hours at 32° C.

[1538] A 10 μg sample of the reaction was analyzed by SDS-PAGE. NovexTris-Glycine 4-12% 1 mm gels were performed and stained using ColloidalBlue as described by Invitrogen. Briefly, samples, 10 μL (10 μg), weremixed with 12 μL sample loading buffer and 1 μL 0.5 M DTT and heated for6 minutes at 85° C. (FIG. 156, lane 4).

[1539] Factor VIIa

[1540] 22. GlycoPEGylation of Recombinant Factor VIIa Produced in BHKCells

[1541] This example sets forth the PEGylation of recombinant Factor VIIamade in BHK cells.

[1542] Preparation of Asialo-Factor VIIa. Recombinant Factor VIIa wasproduced in BHK cells (baby hamster kidney cells). Factor VIIa (14.2 mg)was dissolved at 1 mg/ml in buffer solution (pH 7.4, 0.05 M Tris, 0.15 MNaCl, 0.001 M CaCl₂, 0.05% NaN₃) and was incubated with 300 mU/mLsialidase (Vibrio cholera)-agarose conjugate for 3 days at 32° C. Tomonitor the reaction a small aliquot of the reaction was diluted withthe appropriate buffer and an IEF gel performed according to Invitrogenprocedures (FIG. 157). The mixture was centrifuged at 3,500 rpm and thesupernatant was collected. The resin was washed three times (3×2 mL)with the above buffer solution (pH 7.4, 0.05 M Tris, 0.15 M NaCl, 0.05%NaN₃) and the combined washes were concentrated in a Centricon-Plus-20.The remaining solution was buffer exchanged with 0.05 M Tris (pH 7.4),0.15 M NaCl, 0.05% NaN₃ to a final volume of 14.4 mL.

[1543] Preparation of Factor VIIa-SA-PEG (1 kDa and 10 kDa). Thedesialylation rFactor VIIa solution was split into two equal 7.2 mlsamples. To each sample was added either CMP-SA-5-PEG (1 kDa) (7.4 mg)or CMP-SA-5-PEG (10 kDa) (7.4 mg). ST3Gal3 (1.58U) was added to bothtubes and the reaction mixtures were incubated at 32° C. for 96 hrs. Thereaction was monitored by SDS-PAGE gel using reagents and conditionsdescribed by Invitrogen. When the reaction was complete, the reactionmixture was purified using a Toso Haas TSK-Gel-3000 preparative columnusing PBS buffer (pH 7.1) and collecting fractions based on UVabsorption. The combined fractions containing the product wereconcentrated at 4° C. in Centricon-Plus-20 centrifugal filters(Millipore, Bedford, Mass.) and the concentrated solution reformulatedto yield 1.97 mg (bicinchoninic acid protein assay, BCA assay,Sigma-Aldrich, St. Louis Mo.) of Factor VIIa-PEG. The product of thereaction was analyzed using SDS-PAGE and IEF analysis according to theprocedures and reagents supplied by Invitrogen. Samples were dialyzedagainst water and analyzed by MALDI-TOF. FIG. 158 shows the MALDIresults for native Factor VIIa. FIG. 159 contains the MALDI results forFactor VIIa PEGylated with 1 kDa PEG where peak of Factor VIIa PEGylatedwith 1 KDa PEG is evident. FIG. 160 contains the MALDI results forFactor VIIa PEGylated with 10 kDa PEG where a peak for Factor VIIaPEGylated with 10 kDa PEG is evident. FIG. 161 depicts the SDS-PAGEanalysis of all of the reaction products, where a band for FactorVIIa-SA-PEG (10 kDa) is evident.

[1544] Follicle Stimulating Hormone (FSH)

[1545] 23. GlycoPEGylation of Human Pituitary-Derived FSH

[1546] This example illustrates the assembly of a conjugate of theinvention. Follicle Stimulating Hormone (FSH) is desialylated and thenconjugated with CMP-(sialic acid)-PEG.

[1547] Desialylation of Follicle Stimulating Hormone. FollicleStimulating Hormone (FSH) (Human Pituitary, Calbiochem Cat No. 869001),1 mg, was dissolved in 500 μL 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 5 mMCaCl₂. This solution, 375 μL, was transferred to a small plastic tubeand to it was added 263 mU Neuraminidase II (Vibrio cholerae). Thereaction mixture was shaken gently for 15 hours at 32° C. The reactionmixture was added to N-(p-aminophenyl)oxamic acid-agarose conjugate, 600μL, pre-equilibrated with 50 mM Tris-HCl pH 7.4, 150 mM NaCl and 0.05%NaN₃ and gently rotated 6.5 hours at 4° C. The suspension wascentrifuged for 2 minutes at 14,000 rpm and the supernatant wascollected. The beads were washed 5 times with 0.5 mL of the buffer andall supernatants were pooled. The enzyme solution was dialyzed (7000MWCO) for 15 hours at 4° C. with 2 L of a solution containing 50 mM Tris—HCl pH 7.4, 1 M NaCl, 0.05% NaN₃, and then twice for 4 hours at 4° C.into 50 mM Tris —HCl pH 7.4, 1 M NaCl, 0.05% NaN₃. The solution wasconcentrated to 2 μg/μL by Speed Vac and stored at −20° C. Reactionsamples were analyzed by IEF gels (pH 3-7) (Invitrogen) (FIG. 162).

[1548] Preparation of human pituitary-derived SA-FSH and PEG-SA-FollicleStimulating Hormone. Desialylated FSH (100 μg, 50 μL) and CMP-sialicacid or CMP-SA-PEG (1 kDa or 10 kDa) (0.05 mmol) were dissolved in 13.5μL H₂O (adjusted to pH 8 with NaOH) in 0.5 mL plastic tubes. The tubeswere vortexed briefly and 40 mU ST3Gal3 (36.5 μL) was added (totalvolume 100 μL). The tubes were vortexed again and shaken gently for 24hours at 32° C. The reactions were stopped by freezing at −80° C.Reaction samples of 15 μg were analyzed by SDS-PAGE (FIG. 163), IEF gels(FIG. 164) and MALDI-TOF. Native FSH was also analyzed by SDS-PAGE (FIG.165)

[1549] Analysis of SDS PAGE and IEF Gels of Reaction Products. NovexTris-Glycine 8-16% 1 mm gels for SDS PAGE analysis were purchased fromInvitrogen. 7.5 μL (15 μg) of FSH reaction samples were diluted with 5μL of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% NaN₃ buffer, mixed with15 μL sample loading buffer and 1 μL 9 M μ-mercaptoethanol and heatedfor 6 minutes at 85° C. Gels were run as directed by Invitrogen andstained with Colloidal Blue Stain (Invitrogen).

[1550] FSH samples (15 μg) were diluted with 5 μL Tris buffer and mixedwith 15 μL sample loading buffer (FIG. 162). The samples were thenapplied to Isoelectric Focusing Gels (pH 3-7) (Invitrogen) (FIG. 165).Gels were run and fixed as directed by Invitrogen and then stained withColloidal Blue Stain.

[1551] 24. GlycoPEGylation of Recombinant FSH Produced Recombinantly inCHO Cells

[1552] This example illustrates the assembly of a conjugate of theinvention. Desialylated FSH was conjugated with CMP-(sialic acid)-PEG.

[1553] Preparation of recombinant Asialo-Follicle Stimulation Hormone.Recombinant Follicle Stimulation Hormone (rFSH) produced from CHO wasused in these studies. The 7,500 IU of rFSH was dissolved in 8 mL ofwater. The FSH solution was dialyzed in 50 mM Tris-HCl pH 7.4, 0.15 MNaCl, 5 mM CaCl₂ and concentrated to 500 μL in a Centricon Plus 20centrifugal filter. A portion of this solution (400 μL) (0.8 mg FSH) wastransferred to a small plastic tube and to it was added 275 mUNeuraminidase II (Vibrio cholerae). The reaction mixture was mixed for16 hours at 32° C. The reaction mixture was added to prewashedN-(p-aminophenyl)oxamic acid-agarose conjugate (800 μL) and gentlyrotated for 24 hours at 4° C. The mixture was centrifuged at 10,000 rpmand the supernatant was collected. The beads were washed 3 times with0.6 mL Tris-EDTA buffer, once with 0.4 mL Tris-EDTA buffer and once with0.2 mL of the Tris-EDTA buffer and all supernatants were pooled. Thesupernatant was dialyzed at 4° C. against 2 L of 50 mM Tris —HCl pH 7.4,1 M NaCl, 0.05% NaN₃ and then twice more against 50 mM Tris —HCl pH 7.4,1 M NaCl, 0.05% NaN₃. The dialyzed solution was then concentrated to 420μL in a Centricon Plus 20 centrifugal filter and stored at −20° C.

[1554] Native and desialylated rFSH samples were analyzed by SDS-PAGEand IEF (FIG. 166). Novex Tris-Glycine 8-16% 1 mm gels were purchasedfrom Invitrogen. Samples (7.5 μL, 15 μg) samples were diluted with 5 μLof 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% NaN₃ buffer, mixed with 15μL sample loading buffer and 1 μL 9 M β-mercaptoethanol and heated for 6minutes at 85° C. Gels were run as directed by Invitrogen and stainedwith Colloidal Blue Stain (Invitrogen). Isoelectric Focusing Gels (pH3-7) were purchased from Invitrogen. Samples (7.5 μL, 15 μg) werediluted with 5 μL Tris buffer and mixed with 15 μL sample loadingbuffer. Gels were loaded, run and fixed as directed by Invitrogen. Gelswere stained with Colloidal Blue Stain. Samples of native anddesialylated FSH were also dialyzed against water and analyzed byMALDI-TOF.

[1555] Sialyl-PEGylation of recombinant Follicle Stimulation Hormone.Desialylated FSH (100 μg, 54 μL) and CMP-SA-PEG (1 kDa or 10 kDa) (0.05μmol) were dissolved in 28 μL 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN₃,pH 7.2 in 0.5 mL plastic tubes. The tubes were vortexed briefly and 20mU of ST3Gal3 was added (total volume 100 μL). The tubes were vortexedagain, mixed gently for 24 hours at 32° C. and the reactions stopped byfreezing at −80° C. Samples of this reaction were analyzed as describedabove by SDS-PAGE gels (FIG. 167), IEF gels (FIG. 168) and MALDI-TOF MS.

[1556] MALDI was also performed on the PEGylated rFSH. Duringionization, SA-PEG is eliminated from the N-glycan structure of theglycoprotein. Native FSH gave a peak at 13928; AS-rFSH (13282);resialylated r-FSH (13332); PEG1000-rFSH (13515; 14960 (1); 16455 (2);17796 (3); 19321 (4)); and PEG 10000 (23560 (1); 34790 (2); 45670 (3);and 56760 (4)).

[1557] 25. Pharmacokinetic Study of GlycoPEGylated FSH

[1558] This example sets forth the in vivo testing of thepharmacokinetic properties glycoPEGylated Follicle Stimulating Hormone(FSH) prepared according to the methods of the invention as compared tonon-PEGylated FSH.

[1559] FSH, FSH-SA-PEG (1 kDa) and FSH-SA-PEG (10 kDa) wereradioiodinated using standard conditions (Amersham Biosciences,Arlington Heights, Ill.) and formulated in phosphate buffered salinecontaining 0.1% BSA. After dilution in phosphate buffer to theappropriate concentration, each of the test FSH proteins (0.4 μg, each)was injected intraveneously into female Sprague Dawley rats (250-300 gbody weight) and blood drawn at time points from 0 to 80 hours.Radioactivity in blood samples was analyzed using a gamma counter andthe pharmacokinetics analyzed using standard methods (FIG. 169). FSH wascleared from the blood much more quickly than FSH-PEG (1 kDa), which inturn was clear somewhat more quickly than FSH-PEG (10 kDa).

[1560] 26. Sertoli Cell Bioassay for In Vitro Activity of GlycoPEGylatedFSH

[1561] This example sets forth a bioassay for follicle stimulatinghormone (FSH) activity based on cultured Sertoli cells. This assay isuseful to determine the bioactivity of FSH after glycan remodeling,including glycoconjugation.

[1562] This bioassay is based on the dose-response relationship thatexists between the amount of estradiol produced when FSH, but notlutenizing hormone (LH), is added to cultured Sertoli cells obtainedfrom immature old rats. Exogenous testosterone is converted to17β-estradiol in the presence of FSH.

[1563] Seven to 10 days old Sprague-Dawley rats were used to obtainSertoli cells. After sacrifice, testes were decapsulated and tissue wasdispersed by incubation in collagenase (1 mg/ml), trypsin (1 mg/ml),hyaluronidase (1 mg/ml) and DNases (5 μg/ml) for 5 to 10 min. The tubulefragments settled to the bottom of the flask and were washed in PBS(IX). The tubule fragments were reincubated for 20 min with a mediacontaining the same enzymes: collagenase (1 mg/ml), trypsin (1 mg/ml),hyaluronidase (1 mg/ml) and DNases (5 μg/ml).

[1564] The tubule fragments were homogenized and plated into a 24 wellplate in a serum free media. 5×10⁵ cells were dispersed per well. After48 h incubation at 37° C. and 5% CO₂, fresh media was added to thecells. Composition of the serum free media: DMEM (1 vol), Ham's F10nutrient mixture (1 vol), insulin 1 μg/ml, Transferrin 5 μg/ml, EGF 10ng/ml, T4 20 pg/ml, Hydrocortisone 10⁻⁸ M, Retinoic acid 10⁻⁶ M.

[1565] The stimulation experiment consists of a 24 hour incubation withstandard FSH or samples at 37° C. and 5% CO₂. The mean intra-assaycoefficient of variation is 9% and the mean inter-assay coefficient ofvariation is 11%.

[1566] The 17B-estradiol Elisa Kit DE2000 (R&D Systems, Minneapolis,Minn.) was used to quantify the level of estradiol after incubation withFSH, FSH-SA-PEG (1 kDa) and FSH-SA-PEG (10 kDa).

[1567] The procedure was as follows: 100 μl of Estradiol Standard(provided with kit and prepared as per instructions with kit) or samplewas pipetted into wells of 17B-estradiol Elisa plate(s); 50 μl of17B-estradiol Conjugate (provided with kit, prepared as per instructionswith kit) was added to each well; 50 μl of 17B-estradiol antibodysolution (provided with kit and prepared as per instructions with kit)was added to each well; plates were incubated for 2 hour at roomtemperature at 200 rpm; the liquid was aspirated from each well; thewells were washed 4 times using the washing solution; all the liquid wasremoved from the wells; 200 μL of pNPP Substrate (provided with kit andprepared as per instructions with kit) was added to all wells andincubated for 45 min; 50 μl of Stop solution (provided with kit andprepared as per instructions with kit) was added and the plates wereread it at 405 nm (FIG. 170). While FSH-PEG (10 kDa) exhibited a modeststimulation of Sertoli cells, at 1 μg/ml, FSH-PEG (1 kDa) stimulatedSertoli cells up to 50% more than unPEGylated FSH.

[1568] 27. Steelman-Pohley Bioassay of In Vivo Activity ofGlycoPEGylated FSH

[1569] In this example, the Steelman-Pohley bioassay (Steelman andPohley, 1953, Endocrinology 53:604-615) was used to determine the invivo activity of glycoPEGylated FSH. The Steelman-Pohley assay uses thechange in ovary weight of a rat to measure the in vivo activity of FSHthat is coinjected with human chorionic gonadotropin.

[1570] The Steelman-Pohley bioassay was performed according to theprotocol described in Christin-Maitre et al. (2000, Methods 21:51-57).Seventy female Sprague-Dawley Rats (Charles River Laboratories,Wilmington, Mass.), aged 21 to 22 days, were housed in the testingfacility for at least 5 days before the beginning the assay procedure.Throughout the procedure, the animal room was climate controlled at 18to 26° C., 30 to 70% relative humidity, and 12 hr. artificial light/12hr. dark. All animals were fed Certified Rodent Chow (Harlan Teklad,Madison Wis.) or the equivalent, and water, both ad libitum. Animalprocedures were performed at Calvert Preclinical Services, Inc.(Olyphant, Pa.).

[1571] Recombinant FSH was expressed in CHO cells, purified by standardtechniques and glycoPEGylated with PEG (1 kDa). The rats were dividedinto seven test groups, with ten animals per group. On days −1 and 0,animals of all groups were subcutaneously injected with 20 I.U. of humanchorionic gonadotropin (HCG) in 0.5 ml of 0.9% NaCl. On days 1, 2 and 3,the control animals were subcutaneously injected with a dose of 0.5 mlcontaining 20 I.U. HCG in 0.9% NaCl, while in the other groups, the HCGdose was augmented with either rFSH or rFSH-SA-PEG (1 kDa) at either0.14 μg, 0.4 μg or 1.2 μg per dose. On day 4, the animals wereeuthanized by CO₂ inhalation. The ovaries were removed, trimmed andweighted. The average ovary weight was determined for each group.

[1572]FIG. 171 presents the average ovary weight of the test groups onday 4. The groups receiving HCG alone (control) or the low dose (0.14μg) of either rFSH or rFSH-SA-PEG (1 kDa) had ovary weights that wereroughly equivalent. The groups receiving the medium (0.4 μg) or high(1.2 μg) doses of rFSH or rFSH-SA-PEG (1 kDa) had ovary weights roughlytwice that of the control group. At the medium dose (0.4 μg), theglycoPEGylated rFSH had roughly the same in vivo activity (as determinedby ovary weight) as the unPEGylated rFSH. At the high dose (1.2 μg), theglycoPEGylated rFSH had somewhat higher in vivo activity than theunPEGylated rFSH.

[1573] G-CSF

[1574] 28. GlycoPEGylation of G-CSF Produced in CHO Cells

[1575] Preparation of Asialo-Granulocyte-Colony Stimulation Factor(G-CSF). G-CSF produced in CHO cells is dissolved at 2.5 mg/mL in 50 mMTris 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 5 mM CaCl₂ and concentrated to500 μL in a Centricon Plus 20 centrifugal filter. The solution isincubated with 300 mU/mL Neuraminidase II (Vibrio cholerae) for 16 hoursat 32° C. To monitor the reaction a small aliquot of the reaction isdiluted with the appropriate buffer and a IEF gel performed. Thereaction mixture is then added to prewashed N-(p-aminophenyl)oxamicacid-agarose conjugate (800 μL/mL reaction volume) and the washed beadsgently rotated for 24 hours at 4° C. The mixture is centrifuged at10,000 rpm and the supernatant was collected. The beads are washed 3times with Tris-EDTA buffer, once with 0.4 mL Tris-EDTA buffer and oncewith 0.2 mL of the Tris-EDTA buffer and all supernatants are pooled. Thesupernatant is dialyzed at 4° C. against 50 mM Tris —HCl pH 7.4, 1 MNaCl, 0.05% NaN₃ and then twice more against 50 mM Tris —HCl pH 7.4, 1 MNaCl, 0.05% NaN₃. The dialyzed solution is then concentrated using aCentricon Plus 20 centrifugal filter and stored at −20° C. Theconditions for the IEF gel were run according to the procedures andreagents provided by Invitrogen. Samples of native and desialylatedG-CSF are dialyzed against water and analyzed by MALDI-TOF MS.

[1576] Preparation of G-CSF-(alpha2,3)-Sialyl-PEG. Desialylated G-CSFwas dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN₃,pH 7.2. The solution is incubated with 1 mM CMP-sialic acid-PEG and 0.1U/mL of ST3Gal1 at 32° C. for 2 days. To monitor the incorporation ofsialic acid-PEG, a small aliquot of the reaction hadCMP-SA-PEG-fluorescent ligand added; the label incorporated into thepeptide is separated from the free label by gel filtration on a TosoHaas G3000SW analytical column using PBS buffer (pH 7.1). Thefluorescent label incorporation into the peptide is quantitated using anin-line fluorescent detector. After 2 days, the reaction mixture ispurified using a Toso Haas G3000SW preparative column using PBS buffer(pH 7.1) and collecting fractions based on UV absorption. The product ofthe reaction is analyzed using SDS-PAGE and IEF analysis according tothe procedures and reagents supplied by Invitrogen. Samples of nativeand PEGylated G-CSF are dialyzed against water and analyzed by MALDI-TOFMS.

[1577] Preparation of G-CSF-(alpha2,8)-Sialyl-PEG. G-CSF produced in CHOcells, which contains an alpha2,3-sialylated O-linked glycan, isdissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN₃, pH7.2. The solution is incubated with 1 mM CMP-sialic acid-PEG and 0.1U/mL of CST-II at 32° C. for 2 days. To monitor the incorporation ofsialic acid-PEG, a small aliquot of the reaction hasCMP-SA-PEG-fluorescent ligand added; the label incorporated into thepeptide is separated from the free label by gel filtration on a TosoHaas G3000SW analytical column using PBS buffer (pH 7.1). Thefluorescent label incorporation into the peptide is quantitated using anin-line fluorescent detector. After 2 days, the reaction mixture ispurified using a Toso Haas G3000SW preparative column using PBS buffer(pH 7.1) and collecting fractions based on UV absorption. The product ofthe reaction is analyzed using SDS-PAGE and IEF analysis according tothe procedures and reagents supplied by Invitrogen. Samples of nativeand PEGylated G-CSF are dialyzed against water and analyzed by MALDI-TOFMS.

[1578] Preparation of G-CSF-(alpha2,6)-Sialyl-PEG. G-CSF, containingonly O-linked GalNAc, is dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15M NaCl, 0.05% NaN₃, pH 7.2. The solution is incubated with 1 mMCMP-sialic acid-PEG and 0.1 U/mL of ST6GalNAcI or II at 32° C. for 2days. To monitor the incorporation of sialic acid-PEG, a small aliquotof the reaction has CMP-SA-PEG-fluorescent ligand added; the labelincorporated into the peptide is separated from the free label by gelfiltration on a Toso Haas G3000SW analytical column using PBS buffer (pH7.1). The fluorescent label incorporation into the peptide isquantitated using an in-line fluorescent detector. After 2 days, thereaction mixture is purified using a Toso Haas G3000SW preparativecolumn using PBS buffer (pH 7.1) and collecting fractions based on UVabsorption. The product of the reaction is analyzed using SDS-PAGE andIEF analysis according to the procedures and reagents supplied byInvitrogen. Samples of native and PEGylated G-CSF are dialyzed againstwater and analyzed by MALDI-TOF MS.

[1579] G-CSF produced in CHO cells was treated with Arthrobactersialidase and was then purified by size exclusion on Superdex75 and wastreated with ST3Gal1 or ST3 Gal2 and then with CMP-SA-PEG 20 Kda. Theresulting molecule was purified by ion exchange and gel filtration andanalysis by SDS PAGE demonstrated that the PEGylation was complete. Thisis the first demonstration of glycoPEGylation of an O-linked glycan.

[1580] Glucocerebrosidase

[1581] 29. Glucocerebrosidase-mannose-6-phosphate Produced in CHO Cells

[1582] This example sets forth the procedure to glycoconjugatemannose-6-phosphate to a peptide produced in CHO cells such asglucocerebrosidase.

[1583] Preparation of asialo-glucoceramidase. Glucocerebrosidaseproduced in CHO cells is dissolved at 2.5 mg/mL in 50 mM Tris 50 mMTris-HCl pH 7.4, 0.15 M NaCl, and is incubated with 300 mU/mLsialidase-agarose conjugate for 16 hours at 32° C. To monitor thereaction a small aliquot of the reaction is diluted with the appropriatebuffer and a IEF gel and SDS-PAGE performed according to Invitrogenprocedures. The mixture is centrifuged at 10,000 rpm and the supernatantis collected. The beads are washed 3 times with Tris-EDTA buffer, oncewith 0.4 mL Tris-EDTA buffer, and once with 0.2 mL of the Tris-EDTAbuffer. All supernatants are pooled. The supernatant is dialyzed at 4°C. against 50 mM Tris-HCl pH 7.4, 1 M NaCl, 0.05% NaN₃ and then twicemore against 50 mM Tris-HCl pH 7.4, 1 M NaCl, 0.05% NaN₃. The dialyzedsolution is then concentrated using a Centricon Plus 20 centrifugalfilter. The product of the reaction is analyzed using SDS-PAGE and IEFanalysis according to the procedures and reagents supplied byInvitrogen. Samples are dialyzed against water and analyzed by MALDI-TOFMS.

[1584] Preparation of Glucocerebrosidase-SA-linker-Mannose-6-phosphate(procedure 1). Asialo-glucocerebrosidasefrom above is dissolved at 2.5mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN₃, pH 7.2. The solutionis incubated with 1 mM CMP-sialic acid-linker-Man-6-phosphate and 0.1U/mL of ST3Gal3 at 32° C. for 2 days. To monitor the incorporation ofsialic acid-linker-Man-6-phosphate, a small aliquot of the reaction hadCMP-SA-PEG-fluorescent ligand added; the label incorporated into thepeptide is separated from the free label by gel filtration on a TosoHaas TSK-Gel-3000 analytical column using PBS buffer (pH 7.1). Thefluorescent label incorporation into the peptide is quantitated using anin-line fluorescent detector. When the reaction is complete, thereaction mixture is purified using a Toso Haas TSK-Gel-3000 preparativecolumn using PBS buffer (pH 7.1) and collecting fractions based on UVabsorption. The product of the reaction is analyzed using SDS-PAGE andIEF analysis according to the procedures and reagents supplied byInvitrogen. Samples are dialyzed against water and analyzed by MALDI-TOFMS.

[1585] Preparation of Glucocerebrosidase-SA-linker-Mannose-6-phosphate(procedure 2). Glucocerebrosidase, produced in CHO but incompletelysialylated, is dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl,0.05% NaN₃, pH 7.2. The solution is incubated with 1 mM CMP-sialicacid-linker-Man-6-phosphate and 0.1 U/mL of ST3Gal3 at 32° C. for 2days. To monitor the incorporation of sialicacid-linker-Man-6-phosphate, a small aliquot of the reaction hadCMP-SA-PEG-fluorescent ligand added; the label incorporated into thepeptide is separated from the free label by gel filtration on a TosoHaas TSK-Gel-3000 analytical column using PBS buffer (pH 7.1). Thefluorescent label incorporation into the peptide is quantitated using anin-line fluorescent detector. When the reaction is complete, thereaction mixture is purified using a Toso Haas TSK-Gel-3000 preparativecolumn using PBS buffer (pH 7.1) and collecting fractions based on UVabsorption. The product of the reaction is analyzed using SDS-PAGE andIEF analysis according to the procedures and reagents supplied byInvitrogen. Samples are dialyzed against water and analyzed by MALDI-TOFMS.

[1586] 30. Glucocerebrosidase-Transferrin

[1587] This example sets forth the procedures for the glycoconjugationof proteins, and in particular, transferrin is glycoconjugated toglucocerebrosidase. The GlcNAc-ASN structures are created onglucoceraminidase, and Transferrin-SA-Linker-Gal-UDP is conjugated toGNDF GlcNAc-ASN structures using galactosyltransferase.

[1588] Preparation of GlcNAc-glucocerebrosidase (Cerezyme™). Cerezyme™(glucocerebrosidase) produced in CHO cells is dissolved at 2.5 mg/mL in50 mM Tris 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, and is incubated with 300mU/mL Endo-H-agarose conjugate for 16 hours at 32° C. To monitor thereaction a small aliquot of the reaction is diluted with the appropriatebuffer and a IEF gel and SDS-PAGE performed according to Invitrogenprocedures. The mixture is centrifuged at 10,000 rpm and the supernatantis collected. The beads are washed 3 times with Tris-EDTA buffer, oncewith 0.4 mL Tris-EDTA buffer and once with 0.2 mL of the Tris-EDTAbuffer and all supernatants are pooled. The supernatant is dialyzed at4° C. against 50 mM Tris —HCl pH 7.4, 1 M NaCl, 0.05% NaN₃ and thentwice more against 50 mM Tris —HCl pH 7.4, 1 M NaCl, 0.05% NaN₃. Thedialyzed solution is then concentrated using a Centricon Plus 20centrifugal filter. The product of the reaction is analyzed usingSDS-PAGE and IEF analysis according to the procedures and reagentssupplied by Invitrogen. Samples are dialyzed against water and analyzedby MALDI-TOF MS.

[1589] Preparation of Transferrin-SA-Linker-Gal-glucocerebrosidase.Transferrin-SA-Linker-Gal-UDP from above is dissolved at 2.5 mg/mL in 50mM Tris-HCl, 0.15 M NaCl, 5 mM MnCl₂, 0.05% NaN₃, pH 7.2. The solutionis incubated with 2.5 mg/mL GlcNAc-glucocerebrosidase and 0.1 U/mL ofgalactosyltransferase at 32° C. for 2 days. To monitor the incorporationof glucocerebrosidase, the peptide is separated by gel filtration on aToso Haas G3000SW analytical column using PBS buffer (pH 7.1) and theproduct detected by UV absorption. The reaction mixture is then purifiedusing a Toso Haas G3000SW preparative column using PBS buffer (pH 7.1)collecting fractions based on UV absorption. The product of the reactionis analyzed using SDS-PAGE and IEF analysis according to the proceduresand reagents supplied by Invitrogen. Samples are dialyzed against waterand analyzed by MALDI-TOF MS.

[1590] GM-CSF

[1591] 31. Generation and PEGylation of GlcNAc-ASN Structures: GM-CSFProduced in Saccharomyces

[1592] This example sets forth the preparation of Tissue-type Activatorwith PEGylated GlcNAc-Asn structures.

[1593] Recombinant GM-CSF expressed in yeast is expected to contain 2N-linked and 2 O-linked glycans. The N-linked glycans should be of thebranched mannan type. This recombinant glycoprotein is treated with anendoglycosidase from the group consisting of endoglycosidase H,endoglycosidase-F1, endoglycosidase-F2, endoglycosidase-F3,endoglycosidase-M either alone or in combination with mannosidases I, IIand III to generate GlcNAc nubs on the asparagine (Asn) residues on thepeptide/protein backbone.

[1594] The GlcNAc-Asn structures on the peptide/protein backbone is thenbe modified with galactose or galactose-PEG using UDP-galactose orUDP-galactose-6-PEG, respectively, and a galactosyltransferase such asGalT1. In one case the galactose-PEG is the terminal residue. In thesecond case the galactose is further modified with SA-PEG using aCMP-SA-PEG donor and a sialyltransferase such as ST3GalIII. In anotherembodiment the GlcNAc-Asn structures on the peptide/protein backbone canbe galactosylated and sialylated as described above, and then furthersialylated using CMP-SA-PEG and an α2,8-sialyltranferase such as theenzyme encoded by the Campylobacter jejuni cst-II gene.

[1595] Herceptin™

[1596] 32. Glycoconjugation of Mithramycin to Hercentin™

[1597] This example sets forth the procedures to glycoconjugate a smallmolecule, such as mithramycin to Fc region glycans of an antibodymolecule produced in mammalian cells. Here, the antibody Herceptin™ isused, but one of skill in the art will appreciate that the method can beused with many other antibodies.

[1598] Preparation of Herceptin™-Gal-linker-mithramycin. Herceptin™ isdissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 5 mM MnCl₂, 0.05%NaN₃, pH 7.2. The solution is incubated with 1 mMUDP-galactose-linker-mithramycin and 0.1 U/mL of galactosyltransferaseat 32° C. for 2 days to introduce the mithramycin in the Fc regionglycans. To monitor the incorporation of galactose, a small aliquot ofthe reaction has ¹⁴C-galactose-UDP ligand added; the label incorporatedinto the peptide is separated from the free label by gel filtration on aToso Haas G3000SW analytical column using PBS buffer (pH 7.1). Theradioactive label incorporation into the peptide is quantitated using anin-line radiation detector.

[1599] When the reaction is complete, the reaction mixture is purifiedusing a Toso Haas TSK-Gel-3000 preparative column using PBS buffer (pH7.1) and collecting fractions based on UV absorption. The fractionscontaining product are combined, concentrated, buffer exchanged and thenfreeze-dried. The product of the reaction is analyzed using SDS-PAGE andIEF analysis according to the procedures and reagents supplied byInvitrogen. Samples are dialyzed against water and analyzed by MALDI-TOFMS.

[1600] Interferon α and Interferon β

[1601] 33. GlycoPEGylation of Proteins Expressed in Mammalian or InsectSystems: EPO, Interferon α and Interferon β

[1602] This example sets forth the preparation of PEGylated peptidesthat are expressed in mammalian and insect systems.

[1603] Preparation of acceptor from mammalian expression systems. Thepeptides to be glycoPEGylated using CMP-sialic acid PEG need to haveglycans terminating in galactose. Most peptides from mammalianexpression systems will have terminal sialic acid that first needs to beremoved.

[1604] Sialidase digestion. The peptide is desialylated using asialidase. A typical procedure involves incubating a 1 mg/mL solution ofthe peptide in Tris-buffered saline, pH 7.2, with 5 mM CaCl₂ added, with0.2 U/mL immobilized sialidase from Vibrio cholera (Calbiochem) at 32°C. for 24 hours. Microbial growth can be halted either by sterilefiltration or the inclusion of 0.02% sodium azide. The resin is thenremoved by centrifugation or filtration, and then washed to recoverentrapped peptide. At this point, EDTA may be added to the solution toinhibit any sialidase that has leached from the resin.

[1605] Preparation from insect expression systems. EPO,interferon-alpha, and interferon-beta may also be expressed innon-mammalian systems such as yeast, plants, or insect cells. Thepeptides to be glycoPEGylated using CMP-sialic acid PEG need to haveglycans terminating in galactose. The majority of the N-glycans onpeptides expressed in insect cells, for example, are the trimannosylcore. These glycans are first built out to glycans terminating ingalactose before they are acceptors for sialyltransferase.

[1606] Building acceptor glycans from trimannosyl core. Peptide (1mg/mL) in Tris-buffered saline, pH 7.2, containing 5 mM MnCl₂, 5 mMUDP-glcNAc, 0.05 U/mL GLCNACT I, 0.05 U/mL GLCNACT II, is incubated at32° C. for 24 hours or until the reaction is substantially complete.Microbial growth can be halted either by sterile filtration or theinclusion of 0.02% sodium azide. After buffer exchange to remove UDP andother small molecules, UDP-galactose and MnCl₂ are each added to 5 mM,galactosyltransferase is added to 0.05 U/mL, and is incubated at 32° C.for 24H or until the reaction is substantially complete. Microbialgrowth can be halted either by sterile filtration or the inclusion of0.02% sodium azide. The peptides are then ready for glycoPEGylation.

[1607] Building O-linked glycans. A similar strategy may be employed forinterferon alpha to produce enzymatically the desired O-glycanGal-GalNAc. If necessary, GalNAc linked to serine or threonine can beadded to the peptide using appropriate peptide GalNAc transferases (e.g.GalNAc T1, GalNAc T2, T3, T4, etc.) and UDP-GalNAc. Also, if needed,galactose can be added using galactosyltransferase and UDP-galactose.

[1608] GlycoPEGylation using sialyltransferase. The glycopeptides (1mg/mL) bearing terminal galactose in Tris buffered saline+0.02% sodiumazide are incubated with CMP-SA-PEG (0.75 mM) and 0.4 U/mLsialyltransferase (ST3Gal3 or ST3Gal4 for N-glycans on EPO andinterferon beta; ST3Gal4, or ST3Gal1 for O-glycans on interferon alpha)at 32° C. for 24 hours. Other transferases that may work include the 2,6sialyltransferase from Photobacterium damsella. The acceptor peptideconcentration is most preferably in the range of 0.1 mg/mL up to thesolubility limit of the peptide. The concentration of CMP-SA-PEG shouldbe sufficient for there to be excess over the available sites, but notso high as to cause peptide solubility problems due to the PEG, and mayrange from 50 μM up to 5 mM, and the temperature may range from 2° C. upto 40° C. The time required for complete reaction will depend on thetemperature, the relative amounts of enzyme to acceptor substrate, thedonor substrate concentration, and the pH.

[1609] 34. GlycoPEGylation of Interferon α Produced in CHO Cells

[1610] Preparation of Asialo-Interferon α. Interferon alpha producedfrom CHO cells is dissolved at 2.5 mg/mL in 50 mM Tris 50 mM Tris-HCl pH7.4, 0.15 M NaCl, 5 mM CaCl₂ and concentrated to 500 μL in a CentriconPlus 20 centrifugal filter. The solution is incubated with 300 mU/mLNeuraminidase II (Vibrio cholerae) for 16 hours at 32° C. To monitor thereaction a small aliquot of the reaction is diluted with the appropriatebuffer and a IEF gel performed. The reaction mixture is then added toprewashed N-(p-aminophenyl)oxamic acid-agarose conjugate (800 μL/mLreaction volume) and the washed beads gently rotated for 24 hours at 4°C. The mixture is centrifuged at 10,000 rpm and the supernatant wascollected. The beads are washed 3 times with Tris-EDTA buffer, once with0.4 mL Tris-EDTA buffer and once with 0.2 mL of the Tris-EDTA buffer andall supernatants-were pooled. The supernatant is dialyzed at 4° C.against 50 mM Tris —HCl pH 7.4, 1 M NaCl, 0.05% NaN₃ and then twice moreagainst 50 mM Tris —HCl pH 7.4, 1 M NaCl, 0.05% NaN₃. The dialyzedsolution is then concentrated using a Centricon Plus 20 centrifugalfilter and stored at −20° C. The conditions for the IEF gel are runaccording to the procedures and reagents provided by Invitrogen. Samplesof native and desialylated G-CSF are dialyzed against water and analyzedby MALDI-TOF MS.

[1611] Preparation of Interferon-alpha-(alpha2,3)-Sialyl-PEG.Desialylated interferon-alpha is dissolved at 2.5 mg/mL in 50 mMTris-HCl, 0.15 M NaCl, 0.05% NaN₃, pH 7.2. The solution is incubatedwith 1 mM CMP-sialic acid-PEG and 0.1 U/mL of ST3Gal1 at 32° C. for 2days. To monitor the incorporation of sialic acid-PEG, a small aliquotof the reaction had CMP-SA-PEG-fluorescent ligand added; the labelincorporated into the peptide is separated from the free label by gelfiltration on a Toso Haas G3000SW analytical column using PBS buffer (pH7.1). The fluorescent label incorporation into the peptide isquantitated using an in-line fluorescent detector. After 2 days, thereaction mixture is purified using a Toso Haas G3000SW preparativecolumn using PBS buffer (pH 7.1) and collecting fractions based on UVabsorption. The product of the reaction is analyzed using SDS-PAGE andIEF analysis according to the procedures and reagents supplied byInvitrogen. Samples of native and desialylated Interferon-alpha aredialyzed against water and analyzed by MALDI-TOF MS.

[1612] Preparation of Interferon-alpha-(alpha2,8)-Sialyl-PEG.Interferon-alpha produced in CHO, which contains an alpha2,3-sialylatedO-linked glycan, is dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 MNaCl, 0.05% NaN₃, pH 7.2. The solution is incubated with 1 mM CMP-sialicacid-PEG and 0.1 U/mL of CST-II at 32° C. for 2 days. To monitor theincorporation of sialic acid-PEG, a small aliquot of the reaction hasCMP-SA-PEG-fluorescent ligand added; the label incorporated into thepeptide is separated from the free label by gel filtration on a TosoHaas G3000SW analytical column using PBS buffer (pH 7.1). Thefluorescent label incorporation into the peptide is quantitated using anin-line fluorescent detector. After 2 days, the reaction mixture ispurified using a Toso Haas G3000SW preparative column using PBS buffer(pH 7.1) and collecting fractions based on UV absorption. The product ofthe reaction is analyzed using SDS-PAGE and IEF analysis according tothe procedures and reagents supplied by Invitrogen. Samples of nativeand PEGylated interferon-alpha are dialyzed against water and analyzedby MALDI-TOF MS.

[1613] Preparation of Interferon-alpha-(alpha2,6)-Sialyl-PEG.Interferon-alpha, containing only O-linked GalNAc, was dissolved at 2.5mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN₃, pH 7.2. The solutionis incubated with 1 mM CMP-sialic acid-PEG and 0.1 U/mL of ST6GalNAcI orII at 32° C. for 2 days. To monitor the incorporation of sialicacid-PEG, a small aliquot of the reaction had CMP-SA-PEG-fluorescentligand added; the label incorporated into the peptide is separated fromthe free label by gel filtration on a Toso Haas G3000SW analyticalcolumn using PBS buffer (pH 7.1). The fluorescent label incorporationinto the peptide is quantitated using an in-line fluorescent detector.After 2 days, the reaction mixture is purified using a Toso Haas G3000SWpreparative column using PBS buffer (pH 7.1) and collecting fractionsbased on UV absorption. The product of the reaction is analyzed usingSDS-PAGE and IEF analysis according to the procedures and reagentssupplied by Invitrogen. Samples of native and PEGylated interferon-alphaare dialyzed against water and analyzed by MALDI-TOF MS.

[1614] 35. GlycoPEGylation of Interferon-β-1a With PEG (10 kDa) and PEG(20 kDa)

[1615] This example illustrates a procedure PEGylate Interferon-β witheither PEG (10 kDa) or PEG (20 kDa).

[1616] Briefly, Interferon-β-1a (INF-β) was obtained from Biogen(Avonex™). The IFN-β was first purified by Superdex-75 chromatography.The IFN-β was then desialylated with Vibrio cholerae sialidase. TheINF-β was then PEGylated with SA-PEG (10 kDa) or SA-PEG (20 kDa) andpurified with Superdex-200 chromatography.

[1617] Superdex-75 chromatography purification. INF-β (150 μg) wasapplied to a Superdex-75 column (Amersham Biosciences, ArlingtonHeights, Ill.) and eluted with PBS with 0.5 M NaCl, 0.02 Tween-20, 20 mMhistidine and 10% glycerol. The eluant was monitored for absorbance at280 nm (FIGS. 172A and 172B) and fractions were collected. Peaks 4 and 5were pooled, concentrated in an Amicon Ultra 15 spin filter (Millipore,Billerica, Mass.), and the buffer was exchanged to TBS with 5 mM CaCl₂,0.02% Tween-20, 20 mM histidine and 10% glycerol.

[1618] Sialidase Reaction. The INF-was then desialydated with Vibriocholera salidase (70 mU/ml, CALBIOCHEM®, EMD Biosciences, Inc., SanDiego, Calif.) on agarose in TBS with 5 mM CaCl₂, 0.02% Tween-20, 20 mMhistidine and 10% glycerol. The reaction was carried out at 32° C. for18 hours. The INF-β was removed from the agarose with a 0.22 μm Spin-X™filter (Corning Technology, Inc., Norcross, Ga.). FIG. 173A depicts theMALDI analysis of glycans released from native INF-β. The native INF-βhas many glycoforms containing terminal sialic acid moieties. FIG. 173Bdepicts the MALDI analysis of glycans released from desialylated INF-β.The desialylated INF-β has primarily one glycoform which is bi-antennarywith terminal galactose moieties.

[1619] Lectin Dot-Blot Analysis of Sialylation. Samples of the INF-βfrom the desialidase reaction were dot-blotted onto nitrocellulose andthen blocked with Tris buffered saline (TBS: 0.05M Tris, 0.15M NaCl, pH7.5) and DIG kit (glycan differentiation kit available from Roche #1 210238) blocking buffer. Some of the blots were incubated with Maackiaamurensis agglutinin (MAA) labeled with digoxogenin (DIG) (Roche AppliedScience, Indianapolis, Ill.) to detect αC2,3-sialylation of INF-β. Theseblots were washed with TBS then incubated with anti-digitonin antibodylabeled with alkaline phosphatase, then washed again with TBS anddeveloped with NBT/X-phosphate solution, wherein NBT is 4-nitro bluetetrazolium chloride and X-phosphate is 5-bromo-4-chloro-3indoylphosphate. The left side of FIG. 174 depicts the results of the MAA blotof INF-β after the desialylation reaction. The INF-β is partiallydisialylated, as indicated by the decrease in dot development ascompared to native INF-β in the desialylated samples.

[1620] Other blots were incubated with Erthrina cristagalli lectin (ECL)labeled with biotin (Vector Laboratories, Burlingame, Calif.) to detectexposed galactose residues on INF-β. After incubation with 2.5 μg/mlECL, the blots were washed in TBS and incubated with streptavidinlabeled with alkaline phosphatase. The blots were then washed again anddeveloped. The right side of FIG. 174 depicts the ECL blot afterdevelopment. The increased intensity of the dot of desialylated INF-β ascompared to the native INF-β indicate more exposed galactose moietiesand therefore extensive desialylation.

[1621] PEGylation of Desialylated INF-β with SA-PEG (10 kDa).Desialylated INF-β (0.05 mg/ml) was PEGylated with ST3Gal3 (50 mU/ml)and CMP-SA-PEG (10 kDa) (250 μM) in an appropriate buffer of TBS+5 mMCaCl₂, 0.02% Tween 20, 20 mM histidine, 10% glycerol for 50 hours at 32°C. FIG. 175 depicts the SDS-PAGE analysis of the reaction productsshowing PEGylated INF-β at approximately 98 kDa.

[1622] PEGylation of Desialylated INF-β with SA-PEG (20 kDa).Desialylated INF-β (0.5 mg/ml) was PEGylated with ST3Gal3 (170 mU/ml)and CMP-SA-PEG (20 kDa) in an appropriate buffer of TBS+5 mM CaCl₂,0.02% Tween 20, 20 mM histidine, 10% glycerol for 50 hours at 32° C.FIG. 176 depicts the SDS-PAGE analysis the products of the PEGylationreaction. The PEGylated INF-β has many higher molecular weight bands notfound in the unmodified INF-β indicating extensive PEGylation.

[1623] Superdex-200 Purification of INF-β PEGylated with PEG (10 kDa).The products of the PEGylation reaction were separated on a Superdex-200column (Amersham Biosciences, Arlington Heights, Ill.) in PBS with 0.5NaCl, 0.02 Tween-20, 20 mM histidine and 10% glycerol at 1 ml/min and 30cm/hr flow. The eluant was monitored for absorbance at 280 nm (FIG. 177)and fractions were collected. Peaks 3 and 4 were pooled and concentratedin an Amicon Ultra 15 spin filter.

[1624] Bioassay of INF-β PEGylated With PEG (10 kDa).

[1625] The test is inhibition of the proliferation of the lung carcinomacell line, A549. The A549 cell line are lung carcinoma adherent cellsgrowing in RPMI+10% FBS at 37° C. 5% CO₂. They can be obtained from ATCC# CCL-185. Wash the cells with 10 ml of PBS and remove the PBS. Add 5 mlof trypsin, incubate for 5 minutes at room temperature or 2 minutes at37° C. When the cells are detached resuspend into 25 ml of media andcount the cells. Dilute the cells at a concentration of 10000 cells/mland add 200 ul/well (96 wells plate). Incubate for 4 hours at 37° C. 5%CO₂. Prepare 1 ml of IFN B at a concentration of 0.1 ug/ml. Filter itunder the hood with a 0.2 um filter. Add 100 ul per well (8 replicates=1lane). Incubate for 3 days (do not let the cells go to confluence).Remove 200 ul of media (only 100 ul per well left). Add 25 μl of MTT(Sigma) (5 mg/ml filtered 0.22 μm). Incubate for 4 hours at 37° C. and5% CO₂. Aspirate the media gently and add 100 μl of a mixture ofisopropanol (100 ml and 6N HCl. Aspirate up and down to homogenize thecrystal violet. Read OD 570 nm (remove the background at 630 or 690 nm).

[1626]FIG. 178 depicts the results of the bioassay of the peakscontaining INF-β PEGylated with PEG (10 kDa) as eluted from theSuperdex-200 column.

[1627] Superdex-200 Purification of INF-β PEGylated with PEG (20 kDa).The products of the PEG (20 kDa) PEGylation reaction were separated on aSuperdex-200 column (Amersham Biosciences, Arlington Heights, Ill.) inPBS with 0.5 NaCl, 0.02 Tween-20, 20 mM histidine and 10% glycerol at 1ml/min flow. The eluant was monitored for absorbance at 280 nm (FIG.179) and fractions were collected. Peak 3 contained most of the INF-βPEGylated with PEG (20 kDa).

[1628] Endotoxin Test of INF-β PEGylated With PEG (20 kDa).

[1629] Limulus Lysate Test was performed, BioWhittaker # 50-647U TABLE24 Results of the endotoxin test of INF-β PEGylated with PEG (20 kDa).Concentration INF-β with PEG (20 kDa) 10 EU/ml 0.06 mg/ml  0.16 EU/μgINF-β with PEG (20 kDa)  1 EU/ml 0.07 mg/ml 0.014 EU/μg Native INF-β 40EU/ml  0.1 mg/ml  0.4 EU/μg

[1630] Remicade™

[1631] 36. GlycoPEGylation of Remicade™ Antibody

[1632] This example sets forth the procedure to glycoPEGylate arecombinant antibody molecule by introducing PEG molecules to the Fcregion glycans. Here Remicade™, a TNF-R:IgG Fc region fusion protein, isthe exemplary peptide.

[1633] Preparation of Remicade™-Gal-PEG (10 kDa). Remicade™ is dissolvedat 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 5 mM MnCl₂, 0.05% NaN₃, pH7.2. The solution is incubated with 1 mM UDP-galactose-PEG (10 kDa) and0.1 U/mL of galactosyltransferase at 32° C. for 2 days to introduce thePEG in the Fc region glycans. To monitor the incorporation of galactose,a small aliquot of the reaction has ¹⁴C-galactose-UDP ligand added; thelabel incorporated into the peptide is separated from the free label bygel filtration on a Toso Haas G3000SW analytical column using PBS buffer(pH 7.1). The radioactive label incorporation into the peptide isquantitated using an in-line radiation detector.

[1634] When the reaction is complete, the reaction mixture is purifiedusing a Toso Haas TSK-Gel-3000 preparative column using PBS buffer (pH7.1) and collecting fractions based on UV absorption. The fractionscontaining product are combined, concentrated, buffer exchanged and thenfreeze-dried. The product of the reaction is analyzed using SDS-PAGE andIEF analysis according to the procedures and reagents supplied byInvitrogen. Samples are dialyzed against water and analyzed by MALDI-TOFMS.

[1635] Rituxan™

[1636] 37. Glycoconjugation of Geldanamycin to Rituxan™

[1637] This example sets forth the glycoconjugation of a small molecule,such as geldanamycin, to the Fc region glycans of an antibody producedin CHO cells, such as Rituxan™. Here, the antibody Rituxan™ is used, butone of skill in the art will appreciate that the method can be used withmany other antibodies.

[1638] Preparation of Rituxan™-Gal-linker-geldanamycin. Rituxan™ isdissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 5 mM MnCl₂, 0.05%NaN₃, pH 7.2. The solution is incubated with 1 mMUDP-galactose-linker-geldanamycin and 0.1 U/mL of galactosyltransferaseat 32° C. for 2 days to introduce the geldanamycin in the Fc regionglycans. To monitor the incorporation of galactose, a small aliquot ofthe reaction has ¹⁴C-galactose-UDP ligand added; the label incorporatedinto the peptide is separated from the free label by gel filtration on aToso Haas G3000SW analytical column using PBS buffer (pH 7.1). Theradioactive label incorporation into the peptide is quantitated using anin-line radiation detector.

[1639] When the reaction is complete, the reaction mixture is purifiedusing a Toso Haas TSK-Gel-3000 preparative column using PBS buffer (pH7.1) and collecting fractions based on UV absorption. The fractionscontaining product are combined, concentrated, buffer exchanged and thenfreeze-dried. The product of the reaction is analyzed using SDS-PAGE andIEF analysis according to the procedures and reagents supplied byInvitrogen. Samples are dialyzed against water and analyzed by MALDI-TOFMS.

[1640] Rnase

[1641] 38. Remodeling High Mannose N-Glycans to Hybrid and ComplexN-Glycans: Bovine Pancreatic RNase

[1642] This example sets forth the preparation of bovine pancreas RNasewith hybrid or complex N-glycans. The high mannose N-linked glycans ofthe RNase are enzymatically digested and elaborated to create hybridN-linked glycans. Additionally, the high mannose N-linked glycans of theRNase are enzymatically digested and elaborated to create complexN-linked glycans.

[1643] High mannose structures of N-linked oligosaccharides inglycopeptides can be modified to hybrid or complex forms using thecombination of α-mannosidases and glycosyltransferases. This examplesummarizes the results in such efforts using a simple N-Glycan as amodel substrate.

[1644] Ribonuclease B (RNaseB) purified from bovine pancreas (Sigma) isa glycopeptide consisting of 124 amino acid residues. It has a singlepotential N-glycosylation site modified with high mannose structures.Due to its simplicity and low molecular weight (13.7 kDa to 15.5 kDa),ribonuclease B is a good candidate to demonstrate the feasibility of theN-Glycan remodeling from high mannose structures to hybrid or complexN-linked oligosaccharides. The MALDI-TOF spectrum of RNaseB (FIG. 180A)and HPLC profile for the oligosaccharides cleaved from RNaseB byN-Glycanase (FIG. 180B) indicated that, other than a small portion ofthe non-modified peptide, the majority of N-glycosylation sites of thepeptide are modified with high mannose oligosaccharides consisting of 5to 9 mannose residues.

[1645] Conversion of high mannose N-Glycans to hybrid N-Glycans. Highmannose N-Glycans were converted to hybrid N-Glycans using thecombination of α1,2-mannosidase, GlcNAcT-I (β-1,2-N-acetyl glucosaminyltransferase), GalT-I (β1,4-galactosyltransfease) andα2,3-sialyltransferase /or α2,6-sialyltransferase as shown in FIG. 181.

[1646] As an example, high mannose structures in RNaseB weresuccessfully converted to hybrid structures.

[1647] Man₅GlcNAc₂-R was obtained from Man₅₋₉GlcNAc₂-R catalyzed by asingle α1,2-mannosidase cloned from Trichoderma reesei (FIG. 182). RNaseB (1 g, about 67 μmol) was incubated at 30° C. for 45 hr with 15 mU ofthe recombinant T. reesei α1,2-mannosidase in MES buffer (50 mM, pH 6.5)in a total volume of 10 mL. Man₆₋₉GlcNAc₂-protein structures have beensuccessfully converted to Man₅GlcNAc₂-protein with high efficiency bythe recombinant mannosidase.

[1648] Alternately, Man₅GlcNAc₂—R was obtained from Man₅₋₉GlcNAc₂—Rcatalyzed by a single α1,2-mannosidase purified from Aspergillus saitoi(FIG. 183). RNase B (40 μg, about 2.7 nmol) was incubated at 37° C. for42.5 hr with 25 IU of the commercial A. saitoi α1,2-mannosidase (Glykoor CalBioChem) in NaOAC buffer (100 mM, pH 5.0) in a total volume of 20μl. Man₆₋₉GlcNAc₂-protein structures were successfully converted toMan₅GlcNAc₂-protein by the commercially available mannosidase. However,a new peak corresponding to the GlcNAc-protein appears in the spectrum,indicating the possible contamination of endoglycosidase H in thepreparation. Although several mammalian alpha-mannosidases were requiredto achieve this step, the fungal α1,2-mannosidase was very efficient toremove all α1,2-linked mannose residues.

[1649] GlcNAcT-I then added a GlcNAc residue to the Man₅GlcNAc₂—R (FIG.184). The reaction mixture after the T. reesei α1,2-mannosidase reactioncontaining RNase B (600 μg, about 40 nmol) was incubated withnon-purified recombinant GlcNAcT-1 (34 mU) in MES buffer (50 mM, pH 6.5)containing MnCl₂ (20 mM) and UDP-GlcNAc (5 mM) in a total volume of 400μl. at 37° C. for 42 hr. A GlcNAc residue was quantitatively added toMan₅GlcNAc₂-protein by the recombinant GlcNAcT-I.

[1650] A Gal residue was then added using GalT 1 (FIG. 185). Thereaction mixture after the GnT-I reaction containing RNase B (120 μg,about 8 nmol) was incubated at 37° C. for 20 hr with 3.3 mU of therecombinant GalT-1 in Tris-HCl buffer (100 mM, pH 7.3) containingUDP-Gal (7.5 mM) and MnCl₂ (20 mM) in a total volume of 100 μl. A Galresidue was added to about 98% of the GlcNAc-Man₅GlcNAc₂-protein by therecombinant GalT 1.

[1651] The next step was the addition of a sialic acid using anα2,3-sialyltransferase or an α2,6-sialyltransferase (FIG. 186). As anexample, ST3Gal III, an α2,3-sialyltransferase was used. The reactionmixture after the GalT-1 reaction containing RNase B (13 μg, about 0.87nmol) was incubated at 37° C. for 16 hr with 8.9 mU of recombinantST3Gal III in Tris-HCl buffer (100 mM, pH 7.3) containing CMP-Sialicacid (5 mM) and MnCl₂ (20 mM) in a total volume of 20 μl. A sialic acidresidue was added to about 90% of the Gal-GlcNAc-Man₅GlcNAc₂-protein byrecombinant ST3Gal III using CMP-SA as the donor. The yield can befurther improved by adjusting the reaction conditions.

[1652] For convenience, no purification or dialysis step was requiredafter each reaction described above. More interesting, GalT 1 and ST3GalIII can be combined in a one-pot reaction. Similar yields were obtainedas compared with the separate reactions. The reaction mixture after theGlcNAcT-I reaction containing RNase B (60 μg, about 4 nmol) wasincubated at 37° C. for 20 hr with 1.7 mU of recombinant GalT 1, 9.8 mUof recombinant ST3Gal III in Tris-HCl buffer (100 mM, pH 7.3) containingUDP-Gal (7.5 mM), CMP-sialic acid (5 mM) and MnCl₂ (20 mM) in a totalvolume of 60 μl.

[1653] As shown in FIG. 187, SA-PEG (10 kDa) was successfully added tothe RNaseB. The reaction mixture after the GalT-1 reaction containingRNase B (6.7 μg, about 0.45 nmol) was dialyzed against H₂O for 1 hour atroom temperature and incubated at 37° C. for 15.5 hours with 55 mU ofthe recombinant ST3Gal III in Tris-HCl buffer (50 mM, pH 7.3) containingCMP-SA-PEG (10 kDa) (0.25 mM) and MnCl₂ (20 mM) in a total volume of 20μl. PEG-modified sialic acid residues were successfully added to theGal-GlcNAc-Man₅GlcNAc₂-peptide by the recombinant ST3Gal III. The yieldcan be further improved by adjusting the reaction conditions.

[1654] Conversion of high mannose N-Glycans to complex N-Glycans. Toachieve this conversion, a GlcNAcβ1,2Man₃GlcNAc₂-peptide intermediate isobtained. As shown in FIG. 188, there are at least four feasible routesto carry out the reaction from Man₅GlcNAc₂-peptide to this intermediate:

[1655] Route I: The Man₅GlcNAc₂-peptide produced by the fungal α1,2mannosidase is a substrate of GlcNAc transferase I (GlcNAcT-I, enzyme 2)which adds one GlcNAc. The terminal α1,3- and α1,6-linked mannoseresidues of GlcNAcMansGlcNAc₂-peptide is removed by Golgi α-mannosidaseII (ManII, enzyme 5). This route is a part of the natural pathway forthe processing of N-linked oligosaccharides carried out in higherorganisms.

[1656] Route II: Two mannose residues are first removed by anα-mannosidase (enzyme 6), then a GlcNAc is added by GlcNAcT-I (enzyme2). Other than its natural acceptor Man₅GlcNAc₂R, GlcNAcT-I can alsorecognize Man₃GlcNAc₂R as its substrate and add one GlcNAc to themannose core structure to form GlcNAcMan₃GlcNAc₂-peptide.

[1657] Route III: The α1,6-linked mannose is removed by anα1,6-mannosidase, followed by the addition of GlcNAc by GlcNAcT-I andremoval of the terminal α1,3-linked mannose by an α1,3-mannosidase. Fromthe experimental data obtained, GlcNAcT-1 can recognize thisMan₄GlcNAc₂-peptide as acceptor and add one GlcNAc residue to formGlcNAcMan₄GlcNAc₂-peptide.

[1658] Route IV: Similar to Route III, α1,3-linked mannose is removed byan α1,3-mannosidase, followed by GlcNAcT-I reaction. Then the terminalα1,6-linked mannose can be removed by an α1,6-mannosidase.

[1659] After the function of GlcNAcT-I (responsible for the addition ofthe GlcNAc α1,2-linked to the α1,3-mannose on the mannose core) andGlcNAcT-II (responsible for the addition of a second GlcNAc α1,2-linkedto the α1,6-mannose on the mannose core), the GlcNAc₂Man₃GlcNAc₂-peptidecan be processed by GalT 1 and sialyltransferase to form bi-antennarycomplex N-Glycans. Other GlcNAc transferases such as GlcNAcT-IV,GlcNAcT-V, and/or GlcNAcT-VI (FIG. 188 and FIG. 189) can alsoglycosylate the GlcNAc₂Man₃GlcNAc₂-peptide. Additional glycosylation bythe GalT 1 and sialyltransferases will form multi-antennary complexN-glycans. The enzyme GlcNAcT-III catalyzes the insertion of a bisectingGlcNAc, thus preventing the actions of ManII and subsequent action oftransferases GlcNAcT-II, GlcNAcT-IV and GlcNAcT-V.

[1660] Tissue-Type Plasminogen Activator (TPA)

[1661] 39. Fucosylation of TPA to Create Sialyl Lewis X

[1662] This example sets forth the preparation of Tissue Tissue-typePlasminogen Activator (TPA) with N-linked sialyl Lewis X antigen.

[1663] Sialylation. TPA expressed in mammalian cells will often containa majority of the glycans terminating in sialic acid, but to ensurecomplete sialylation, it would be beneficial to first perform an invitro sialylation. TPA in a suitable buffer (most preferably between pH5.5 and 9, for example Tris buffered saline, pH 7.2) is incubated withCMP sialic acid and sialyltransferase for a time sufficient to convertany glycans lacking sialic acid to sialylated species. Typicalconditions would be 1 mg/mL TPA, 3 mM CMP sialic acid, 0.02 U/mLST3Gal3, 32° C. for 24 hours. Microbial growth can be halted either bysterile filtration or the inclusion of 0.02% sodium azide. The TPAconcentration is most preferably in the range 0.1 mg/mL up to thesolubility limit of the peptide. The concentration of CMP-SA should besufficient for there to be excess over the available sites, and mightrange from 50 μM up to 50 mM, and the temperature from 2° C. up to 40°C. The time required for complete reaction will depend on thetemperature, the relative amounts of enzyme to acceptor substrate, thedonor substrate concentration, and the pH. Other sialyltransferases thatmay be capable of adding sialic acid in 2,3 linkage include ST3Gal4;microbial transferases could also be used.

[1664] Fucosylation. Typical conditions for fucosylation would be 1mg/mL TPA, 3 mM GDP-fucose, 0.02 U/mL FTVI, 5 mM MnCl₂, 32° C. for 24Hin Tris buffered saline. Microbial growth can be halted either bysterile filtration or the inclusion of 0.02% sodium azide. The TPAconcentration is most preferably in the range 0.1 mg/mL up to thesolubility limit of the peptide. The concentration of GDP-fucose shouldbe sufficient for there to be excess over the available sites, and mightrange from 50 μM up to 50 mM, and the temperature from 2° C. up to 40°C. The time required for complete reaction will depend on thetemperature, the relative amounts of enzyme to acceptor substrate, thedonor substrate concentration, and the pH. Other fucosyltransferasesthat may be capable of making sialyl Lewis x include FTVII, FTV, FrIII,as well as microbial transferases could also be used.

[1665] 40. Trimming of High Mannose to Tri-Mannose Core Structure:Tissue-Type Plasminogen Activator Produced in CHO

[1666] This example sets forth the preparation of Tissue-typePlasminogen Activator with a trimannose core by trimming back from ahigh mannose glycan.

[1667] Tissue-type plasminogen activator (TPA) is currently produced inChinese Hamster Ovary (CHO) cells and contains a low amount of highmannose N-linked oligosaccharide. The mannoses can be trimmed down usinga variety of the specific mannosidases. The first step is to generateMan5GlcNAc2(Fuc0-1) from Man9GlcNAc2(Fuc0-1). This can be done usingmannosidase I. Then either GlcNAcT1 (GlcNAc transferase I) is used tomake GlcNAc1Man5GlcNAc2(Fuc0-1) or Mannosidase III is used to makeMan3GlcNAc2(Fuc0-1). From Man3GlcNAc2(Fuc0-1),GlcNAc1Man3GlcNAc2(Fuc0-1) can be produced using GlcNAcT1 or fromGlcNAc1Man5GlcNAc2(Fuc0-1), GlcNAc1Man3GlcNAc2(Fuc0-1) can be producedusing Mannosidase II. GlcNAc1Man3GlcNAc2(Fuc0-1) is then converted intoGlcNAc2Man3GlcNAc2(Fuc0-1) using GlcNAcTransferase II (GlcNAcTII). Thetwo terminal GlcNAc residues are then galactosylated using GalTI andthen sialylated with SA-PEG using ST3GalIII.

[1668] Conversely, TPA can be produce in yeast or fungal systems.Similar processing would be required for fungal derived material.

[1669] 41. Generation and PEGylation of GlcNAc-ASN Structures: TPAProduced in Yeast

[1670] This example sets forth the preparation of PEGylated GlcNAc-Asnstructures on a peptide such as TPA expressed in yeast.

[1671] Yeast expression is expected to result in a TPA which contains asingle N-linked mannan-type structure. This recombinant glycoprotein isfirst treated with endoglycosidase H to generate GlcNAc structures onthe asparagine (Asn) residues on the peptide.

[1672] The GlcNAc-Asn structures on the peptide/protein backbone arethen be modified with galactose or galactose-PEG using UDP-galactose orUDP-galactose-6-PEG, respectively, and a galactosyltransferase such asGalTI. In one case, the galactose-PEG is the terminal residue. In thesecond case, the galactose is further modified with SA-PEG using aCMP-SA-PEG donor and a sialyltransferase such as ST3GalIII. In anotherembodiment, the GlcNAc-Asn structures on the peptide/protein backbonemay be galactosylated and sialylated as described above, and thenfurther sialylated using CMP-SA-PEG and an α2,8-sialyltransferase suchas the enzyme encoded by the Campylobacter jejuni cst-II gene.

[1673] Transferrin

[1674] 42. GlycoPEGylation of Transferrin

[1675] This example sets forth the preparation of asialotransferrin andits sialylation with PEG-CMP-sialic acid.

[1676] Preparation of Asialo-transferrin. Human-derivedholo-Transferrin, (10 mg) was dissolved in 500 μL of 50 mM NaOAc, 5 mMCaCl₂, pH 5.5. To this solution was added 500 mU Neuraminidase II(Vibrio cholerae) and the reaction mixture was shaken gently for 20.5hours at 37° C. The reaction mixture was added to the prewashedN-(p-aminophenyl)oxamic acid-agarose conjugate (600 μL) and the washedbeads gently rotated for 24 hours at 4° C. The mixture was centrifugedat 10,000 rpm and the supernatant was collected. The reaction mixturewas adjusted to 5 mM EDTA by addition of 100 μL of 30 mM EDTA to thewashed beads, which were gently rotated for 20 hours at 4° C. Thesuspension was centrifuged for 2 minutes at 10,000 rpm and thesupernatant was collected. The beads were washed 5 times with 0.35 mL of50 mM NaOAc, 5 mM CaCl₂, 5 mM EDTA, pH 5.5 and all supernatants werepooled. The enzyme solution was dialyzed twice at 4° C. into 15 mMTris-HCl, 1 M NaCl, pH 7.4. 0.3 mL of the transferrin solution (3.3 mLtotal) was removed and dialyzed twice against water. The remainder wasdialyzed twice more at 4° C. against phosphate buffered saline. Thedialyzed solution was stored at −20° C. Protein samples were analyzed byIEF Electrophoresis. Samples (9 μL, 25 μg) were diluted with 16 μL Trisbuffer and mixed with 25 μL of the sample loading buffer and applied toIsoelectric Focusing Gels (pH 3-7). Gels were run and fixed usingstandard procedures. Gels were stained with Colloidal Blue Stain.

[1677] Sialyl-PEGylation of asialo-Transferrin. Desialylated transferrin(250 μg) and CMP-sialic acid or CMP-SA-PEG (1 kDa or 10 kDa)(0.05 μmol)were dissolved in 69 μL 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN₃, pH 7.2in 1.5 mL plastic tubes. The tubes were vortexed briefly and 100 mUST3Gal3 (90 μL) were added (total volume 250 μL). The tubes werevortexed again and mixed gently for 24 hours at 32° C. The reactionswere stopped by freezing at −80° C. Novex Tris-Glycine 8-16% 1 mm gelswere used for SDS PAGE analysis (FIG. 190). Samples (25 μL, 25 μg) weremixed with 25 μL of sample loading buffer and 0.4 μL ofβ-mercaptoethanol and heated for 6 minutes at 85° C. Gels were run usingstandard conditions and stained with Colloidal Blue Stain. IEF gels werealso performed as described above FIG. 191). Samples were also dialyzedagainst water analyzed by MALDI-TOF.

[1678] Results. MALDI was also performed. Native transferrin (78729);asialotransferrin (78197); resialylated transferrin (79626/80703); withSA-PEG 1k (79037 (1); 80961 (2); 82535 (3); 84778 (4)); with SA-PEG 5k(90003 (2); 96117 (3); 96117 (4)); with SA-PEG 10k (100336 (2); 111421(3); 122510 (4)).

[1679] 43. Transferrin-GDNF

[1680] This example sets forth the procedures for the glycoconjugationof proteins, and in particular, transferrin is glycoconjugated to GDNF.Transferrin-SA-Linker-Gal-UDP is prepared from transferrin. Thegalactose residue is removed from GNDF glycans, andTransferrin-SA-Linker-Gal-UDP is conjugated to GNDF glycans using agalactosyltransferase.

[1681] Preparation of agalacto-GDNF. GDNF produced in NSO cells (NSOmurine myeloma cells) is dissolved at 2.5 mg/mL in 50 mM Tris 50 mMTris-HCl pH 7.4, 0.15 M NaCl, and is incubated with 300 mU/mLbeta-galactosidase-agarose conjugate for 16 hours at 32° C. To monitorthe reaction a small aliquot of the reaction is diluted with theappropriate buffer and a IEF gel performed according to Invitrogenprocedures. The mixture is centrifuged at 10,000 rpm and the supernatantis collected. The supernatant is dialyzed at 4° C. against 50 mM Tris—HCl pH 7.4, 1 M NaCl, 0.05% NaN₃ and then twice more against 50 mM Tris—HCl pH 7.4, 1 M NaCl, 0.05% NaN₃. The dialyzed solution is thenconcentrated using a Centricon Plus 20 centrifugal filter and stored at−20° C. The conditions for the IEF gel are run according to theprocedures and reagents provided by Invitrogen. Samples are dialyzedagainst water and analyzed by MALDI-TOF MS.

[1682] Preparation of Transferrin-SA-Linker-Gal-UDP. Asialo-transferrinis dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN₃, pH7.2. The solution is incubated with CMP-sialic acid-linker-Gal-UDP(molar amount to add 1 molar equivalent of nucleotide sugar totransferrin) and 0.1 U/mL of ST3Gal3 at 32° C. for 2 days. To monitorthe incorporation of sialic acid, a small aliquot of the reaction has¹⁴C-SA-UDP ligand added; the label incorporated into the peptide isseparated from the free label by gel filtration on a Toso Haas G3000SWanalytical column using PBS buffer (pH 7.1). The radioactive labelincorporation into the peptide is quantitated using an in-line radiationdetector.

[1683] The solution is incubated with 5 mM CMP-sialic acid and 0.1 U/mLof ST3Gal3 (to cap any unreacted transferrin glycans) at 32° C. for 2days. The incorporation into the peptide is quantitated using an in-lineUV detector. After 2 days, the reaction mixture is purified using a TosoHaas G3000SW preparative column using PBS buffer (pH 7.1) and collectingfractions based on UV absorption. The product of the reaction isanalyzed using SDS-PAGE and IEF analysis according to the procedures andreagents supplied by Invitrogen. Samples are dialyzed against water andanalyzed by MALDI-TOF MS.

[1684] Preparation of Transferrin-SA-Linker-Gal-GDNF. Thetransferrin-SA-Linker-Gal-UDP prepared as described above is dissolvedat 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 5 mM MnCl₂, 0.05% NaN₃, pH7.2. The solution is incubated with 2.5 mg/mL agalacto-GDNF and 0.1 U/mLof galactosyltransferase at 32° C. for 2 days. To monitor theincorporation of galactose, a small aliquot of the reaction has¹⁴C-galactose-UDP ligand added; the label incorporated into the peptideis separated from the free label by gel filtration on a Toso HaasG3000SW analytical column using PBS buffer (pH 7.1). The radioactivelabel incorporation into the peptide is quantitated using an in-lineradiation detector.

[1685] When the reaction is complete, the solution is incubated with 5mM UDP-Gal and 0.1 U/mL of galactosyltransferase (to cap any unreactedtransferrin glycans) at 32° C. for 2 days followed by addition of 5 mMCMP-SA and 0.1 U/mL of ST3Gal3. After 2 additional days, the reactionmixture is purified using a Toso Haas G3000SW preparative column usingPBS buffer (pH 7.1) collecting fractions based on UV absorption. Theproduct of the reaction is analyzed using SDS-PAGE and IEF analysisaccording to the procedures and reagents supplied by Invitrogen. Samplesare dialyzed against water and analyzed by MALDI-TOF MS.

[1686] The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

[1687] While this invention has been disclosed with reference tospecific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1 75 1 525 DNA Homo sapiens 1 acccccctgg gccctgccag ctccctgccccagagcttcc tgctcaagtg cttagagcaa 60 gtgaggaaga tccagggcga tggcgcagcgctccaggaga agctgtgtgc cacctacaag 120 ctgtgccacc ccgaggagct ggtgctgctcggacactctc tgggcatccc ctgggctccc 180 ctgagcagct gccccagcca ggccctgcagctggcaggct gcttgagcca actccatagc 240 ggccttttcc tctaccaggg gctcctgcaggccctggaag ggatctcccc cgagttgggt 300 cccaccttgg acacactgca gctggacgtcgccgactttg ccaccaccat ctggcagcag 360 atggaagaac tgggaatggc ccctgccctgcagcccaccc agggtgccat gccggccttc 420 gcctctgctt tccagcgccg ggcaggaggggtcctggttg cctcccatct gcagagcttc 480 ctggaggtgt cgtaccgcgt tctacgccaccttgcccagc cctga 525 2 174 PRT Homo sapiens 2 Thr Pro Leu Gly Pro AlaSer Ser Leu Pro Gln Ser Phe Leu Leu Lys 1 5 10 15 Cys Leu Glu Gln ValArg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln 20 25 30 Glu Lys Leu Cys AlaThr Tyr Lys Leu Cys His Pro Glu Glu Leu Val 35 40 45 Leu Leu Gly His SerLeu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys 50 55 60 Pro Ser Gln Ala LeuGln Leu Ala Gly Cys Leu Ser Gln Leu His Ser 65 70 75 80 Gly Leu Phe LeuTyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser 85 90 95 Pro Glu Leu GlyPro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp 100 105 110 Phe Ala ThrThr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro 115 120 125 Ala LeuGln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe 130 135 140 GlnArg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe 145 150 155160 Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 165 170 31733 DNA Homo sapiens 3 gcgcctctta tgtacccaca aaaatctatt ttcaaaaaagttgctctaag aatatagtta 60 tcaagttaag taaaatgtca atagcctttt aatttaatttttaattgttt tatcattctt 120 tgcaataata aaacattaac tttatacttt ttaatttaatgtatagaata gagatataca 180 taggatatgt aaatagatac acagtgtata tgtgattaaaatataatggg agattcaatc 240 agaaaaaagt ttctaaaaag gctctggggt aaaagaggaaggaaacaata atgaaaaaaa 300 tgtggtgaga aaaacagctg aaaacccatg taaagagtgtataaagaaag caaaaagaga 360 agtagaaagt aacacagggg catttggaaa atgtaaacgagtatgttccc tatttaaggc 420 taggcacaaa gcaaggtctt cagagaacct ggagcctaaggtttaggctc acccatttca 480 accagtctag cagcatctgc aacatctaca atggccttgacctttgcttt actggtggcc 540 ctcctggtgc tcagctgcaa gtcaagctgc tctgtgggctgtgatctgcc tcaaacccac 600 agcctgggta gcaggaggac cttgatgctc ctggcacagatgaggagaat ctctcttttc 660 tcctgcttga aggacagaca tgactttgga tttccccaggaggagtttgg caaccagttc 720 caaaaggctg aaaccatccc tgtcctccat gagatgatccagcagatctt caatctcttc 780 agcacaaagg actcatctgc tgcttgggat gagaccctcctagacaaatt ctacactgaa 840 ctctaccagc agctgaatga cctggaagcc tgtgtgatacagggggtggg ggtgacagag 900 actcccctga tgaaggagga ctccattctg gctgtgaggaaatacttcca aagaatcact 960 ctctatctga aagagaagaa atacagccct tgtgcctgggaggttgtcag agcagaaatc 1020 atgagatctt tttctttgtc aacaaacttg caagaaagtttaagaagtaa ggaatgaaaa 1080 ctggttcaac atggaaatga ttttcattga ttcgtatgccagctcacctt tttatgatct 1140 gccatttcaa agactcatgt ttctgctatg accatgacacgatttaaatc ttttcaaatg 1200 tttttaggag tattaatcaa cattgtattc agctcttaaggcactagtcc cttacagagg 1260 accatgctga ctgatccatt atctatttaa atatttttaaaatattattt atttaactat 1320 ttataaaaca acttattttt gttcatatta tgtcatgtgcacctttgcac agtggttaat 1380 gtaataaaat gtgttctttg tatttggtaa atttattttgtgttgttcat tgaacttttg 1440 ctatggaact tttgtacttg tttattcttt aaaatgaaattccaagccta attgtgcaac 1500 ctgattacag aataactggt acacttcatt tgtccatcaatattatattc aagatataag 1560 taaaaataaa ctttctgtaa accaagttgt atgttgtactcaagataaca gggtgaacct 1620 aacaaataca attctgctct cttgtgtatt tgatttttgtatgaaaaaaa ctaaaaatgg 1680 taatcatact taattatcag ttatggtaaa tggtatgaagagaagaagga acg 1733 4 188 PRT Homo sapiens 4 Met Ala Leu Thr Phe Ala LeuLeu Val Ala Leu Leu Val Leu Ser Cys 1 5 10 15 Lys Ser Ser Cys Ser ValGly Cys Asp Leu Pro Gln Thr His Ser Leu 20 25 30 Gly Ser Arg Arg Thr LeuMet Leu Leu Ala Gln Met Arg Arg Ile Ser 35 40 45 Leu Phe Ser Cys Leu LysAsp Arg His Asp Phe Gly Phe Pro Gln Glu 50 55 60 Glu Phe Gly Asn Gln PheGln Lys Ala Glu Thr Ile Pro Val Leu His 65 70 75 80 Glu Met Ile Gln GlnIle Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser 85 90 95 Ala Ala Trp Asp GluThr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr 100 105 110 Gln Gln Leu AsnAsp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val 115 120 125 Thr Glu ThrPro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys 130 135 140 Tyr PheGln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro 145 150 155 160Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu 165 170175 Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu 180 185 5 757 DNAHomo sapiens 5 atgaccaaca agtgtctcct ccaaattgct ctcctgttgt gcttctccactacagctctt 60 tccatgagct acaacttgct tggattccta caaagaagca gcaattttcagtgtcagaag 120 ctcctgtggc aattgaatgg gaggcttgaa tattgcctca aggacaggatgaactttgac 180 atccctgagg agattaagca gctgcagcag ttccagaagg aggacgccgcattgaccatc 240 tatgagatgc tccagaacat ctttgctatt ttcagacaag attcatctagcactggctgg 300 aatgagacta ttgttgagaa cctcctggct aatgtctatc atcagataaaccatctgaag 360 acagtcctgg aagaaaaact ggagaaagaa gattttacca ggggaaaactcatgagcagt 420 ctgcacctga aaagatatta tgggaggatt ctgcattacc tgaaggccaaggagtacagt 480 cactgtgcct ggaccatagt cagagtggaa atcctaagga acttttacttcattaacaga 540 cttacaggtt acctccgaaa ctgaagatct cctagcctgt ccctctgggactggacaatt 600 gcttcaagca ttcttcaacc agcagatgct gtttaagtga ctgatggctaatgtactgca 660 aatgaaagga cactagaaga ttttgaaatt tttattaaat tatgagttatttttatttat 720 ttaaatttta ttttggaaaa taaattattt ttggtgc 757 6 187 PRTHomo sapiens 6 Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu CysPhe Ser 1 5 10 15 Thr Thr Ala Leu Ser Met Ser Tyr Asn Leu Leu Gly PheLeu Gln Arg 20 25 30 Ser Ser Asn Phe Gln Cys Gln Lys Leu Leu Trp Gln LeuAsn Gly Arg 35 40 45 Leu Glu Tyr Cys Leu Lys Asp Arg Met Asn Phe Asp IlePro Glu Glu 50 55 60 Ile Lys Gln Leu Gln Gln Phe Gln Lys Glu Asp Ala AlaLeu Thr Ile 65 70 75 80 Tyr Glu Met Leu Gln Asn Ile Phe Ala Ile Phe ArgGln Asp Ser Ser 85 90 95 Ser Thr Gly Trp Asn Glu Thr Ile Val Glu Asn LeuLeu Ala Asn Val 100 105 110 Tyr His Gln Ile Asn His Leu Lys Thr Val LeuGlu Glu Lys Leu Glu 115 120 125 Lys Glu Asp Phe Thr Arg Gly Lys Leu MetSer Ser Leu His Leu Lys 130 135 140 Arg Tyr Tyr Gly Arg Ile Leu His TyrLeu Lys Ala Lys Glu Tyr Ser 145 150 155 160 His Cys Ala Trp Thr Ile ValArg Val Glu Ile Leu Arg Asn Phe Tyr 165 170 175 Phe Ile Asn Arg Leu ThrGly Tyr Leu Arg Asn 180 185 7 1332 DNA Homo sapiens 7 atggtctcccaggccctcag gctcctctgc cttctgcttg ggcttcaggg ctgcctggct 60 gcagtcttcgtaacccagga ggaagcccac ggcgtcctgc accggcgccg gcgcgccaac 120 gcgttcctggaggagctgcg gccgggctcc ctggagaggg agtgcaagga ggagcagtgc 180 tccttcgaggaggcccggga gatcttcaag gacgcggaga ggacgaagct gttctggatt 240 tcttacagtgatggggacca gtgtgcctca agtccatgcc agaatggggg ctcctgcaag 300 gaccagctccagtcctatat ctgcttctgc ctccctgcct tcgagggccg gaactgtgag 360 acgcacaaggatgaccagct gatctgtgtg aacgagaacg gcggctgtga gcagtactgc 420 agtgaccacacgggcaccaa gcgctcctgt cggtgccacg aggggtactc tctgctggca 480 gacggggtgtcctgcacacc cacagttgaa tatccatgtg gaaaaatacc tattctagaa 540 aaaagaaatgccagcaaacc ccaaggccga attgtggggg gcaaggtgtg ccccaaaggg 600 gagtgtccatggcaggtcct gttgttggtg aatggagctc agttgtgtgg ggggaccctg 660 atcaacaccatctgggtggt ctccgcggcc cactgtttcg acaaaatcaa gaactggagg 720 aacctgatcgcggtgctggg cgagcacgac ctcagcgagc acgacgggga tgagcagagc 780 cggcgggtggcgcaggtcat catccccagc acgtacgtcc cgggcaccac caaccacgac 840 atcgcgctgctccgcctgca ccagcccgtg gtcctcactg accatgtggt gcccctctgc 900 ctgcccgaacggacgttctc tgagaggacg ctggccttcg tgcgcttctc attggtcagc 960 ggctggggccagctgctgga ccgtggcgcc acggccctgg agctcatggt gctcaacgtg 1020 ccccggctgatgacccagga ctgcctgcag cagtcacgga aggtgggaga ctccccaaat 1080 atcacggagtacatgttctg tgccggctac tcggatggca gcaaggactc ctgcaagggg 1140 gacagtggaggcccacatgc cacccactac cggggcacgt ggtacctgac gggcatcgtc 1200 agctggggccagggctgcgc aaccgtgggc cactttgggg tgtacaccag ggtctcccag 1260 tacatcgagtggctgcaaaa gctcatgcgc tcagagccac gcccaggagt cctcctgcga 1320 gccccatttccc 1332 8 444 PRT Homo sapiens 8 Met Val Ser Gln Ala Leu Arg Leu Leu CysLeu Leu Leu Gly Leu Gln 1 5 10 15 Gly Cys Leu Ala Ala Val Phe Val ThrGln Glu Glu Ala His Gly Val 20 25 30 Leu His Arg Arg Arg Arg Ala Asn AlaPhe Leu Glu Glu Leu Arg Pro 35 40 45 Gly Ser Leu Glu Arg Glu Cys Lys GluGlu Gln Cys Ser Phe Glu Glu 50 55 60 Ala Arg Glu Ile Phe Lys Asp Ala GluArg Thr Lys Leu Phe Trp Ile 65 70 75 80 Ser Tyr Ser Asp Gly Asp Gln CysAla Ser Ser Pro Cys Gln Asn Gly 85 90 95 Gly Ser Cys Lys Asp Gln Leu GlnSer Tyr Ile Cys Phe Cys Leu Pro 100 105 110 Ala Phe Glu Gly Arg Asn CysGlu Thr His Lys Asp Asp Gln Leu Ile 115 120 125 Cys Val Asn Glu Asn GlyGly Cys Glu Gln Tyr Cys Ser Asp His Thr 130 135 140 Gly Thr Lys Arg SerCys Arg Cys His Glu Gly Tyr Ser Leu Leu Ala 145 150 155 160 Asp Gly ValSer Cys Thr Pro Thr Val Glu Tyr Pro Cys Gly Lys Ile 165 170 175 Pro IleLeu Glu Lys Arg Asn Ala Ser Lys Pro Gln Gly Arg Ile Val 180 185 190 GlyGly Lys Val Cys Pro Lys Gly Glu Cys Pro Trp Gln Val Leu Leu 195 200 205Leu Val Asn Gly Ala Gln Leu Cys Gly Gly Thr Leu Ile Asn Thr Ile 210 215220 Trp Val Val Ser Ala Ala His Cys Phe Asp Lys Ile Lys Asn Trp Arg 225230 235 240 Asn Leu Ile Ala Val Leu Gly Glu His Asp Leu Ser Glu His AspGly 245 250 255 Asp Glu Gln Ser Arg Arg Val Ala Gln Val Ile Ile Pro SerThr Tyr 260 265 270 Val Pro Gly Thr Thr Asn His Asp Ile Ala Leu Leu ArgLeu His Gln 275 280 285 Pro Val Val Leu Thr Asp His Val Val Pro Leu CysLeu Pro Glu Arg 290 295 300 Thr Phe Ser Glu Arg Thr Leu Ala Phe Val ArgPhe Ser Leu Val Ser 305 310 315 320 Gly Trp Gly Gln Leu Leu Asp Arg GlyAla Thr Ala Leu Glu Leu Met 325 330 335 Val Leu Asn Val Pro Arg Leu MetThr Gln Asp Cys Leu Gln Gln Ser 340 345 350 Arg Lys Val Gly Asp Ser ProAsn Ile Thr Glu Tyr Met Phe Cys Ala 355 360 365 Gly Tyr Ser Asp Gly SerLys Asp Ser Cys Lys Gly Asp Ser Gly Gly 370 375 380 Pro His Ala Thr HisTyr Arg Gly Thr Trp Tyr Leu Thr Gly Ile Val 385 390 395 400 Ser Trp GlyGln Gly Cys Ala Thr Val Gly His Phe Gly Val Tyr Thr 405 410 415 Arg ValSer Gln Tyr Ile Glu Trp Leu Gln Lys Leu Met Arg Ser Glu 420 425 430 ProArg Pro Gly Val Leu Leu Arg Ala Pro Phe Pro 435 440 9 1437 DNA Homosapiens 9 atgcagcgcg tgaacatgat catggcagaa tcaccaagcc tcatcaccatctgcctttta 60 ggatatctac tcagtgctga atgtacagtt tttcttgatc atgaaaacgccaacaaaatt 120 ctgaatcggc caaagaggta taattcaggt aaattggaag agtttgttcaagggaacctt 180 gagagagaat gtatggaaga aaagtgtagt tttgaagaac cacgagaagtttttgaaaac 240 actgaaaaga caactgaatt ttggaagcag tatgttgatg gagatcagtgtgagtccaat 300 ccatgtttaa atggcggcag ttgcaaggat gacattaatt cctatgaatgttggtgtccc 360 tttggatttg aaggaaagaa ctgtgaatta gatgtaacat gtaacattaagaatggcaga 420 tgcgagcagt tttgtaaaaa tagtgctgat aacaaggtgg tttgctcctgtactgaggga 480 tatcgacttg cagaaaacca gaagtcctgt gaaccagcag tgccatttccatgtggaaga 540 gtttctgttt cacaaacttc taagctcacc cgtgctgagg ctgtttttcctgatgtggac 600 tatgtaaatc ctactgaagc tgaaaccatt ttggataaca tcactcaaggcacccaatca 660 tttaatgact tcactcgggt tgttggtgga gaagatgcca aaccaggtcaattcccttgg 720 caggttgttt tgaatggtaa agttgatgca ttctgtggag gctctatcgttaatgaaaaa 780 tggattgtaa ctgctgccca ctgtgttgaa actggtgtta aaattacagttgtcgcaggt 840 gaacataata ttgaggagac agaacataca gagcaaaagc gaaatgtgattcgagcaatt 900 attcctcacc acaactacaa tgcagctatt aataagtaca accatgacattgcccttctg 960 gaactggacg aacccttagt gctaaacagc tacgttacac ctatttgcattgctgacaag 1020 gaatacacga acatcttcct caaatttgga tctggctatg taagtggctgggcaagagtc 1080 ttccacaaag ggagatcagc tttagttctt cagtacctta gagttccacttgttgaccga 1140 gccacatgtc ttcgatctac aaagttcacc atctataaca acatgttctgtgctggcttc 1200 catgaaggag gtagagattc atgtcaagga gatagtgggg gaccccatgttactgaagtg 1260 gaagggacca gtttcttaac tggaattatt agctggggtg aagagtgtgcaatgaaaggc 1320 aaatatggaa tatataccaa ggtatcccgg tatgtcaact ggattaaggaaaaaacaaag 1380 ctcacttaat gaaagatgga tttccaaggt taattcattg gaattgaaaattaacag 1437 10 462 PRT Homo sapiens 10 Met Gln Arg Val Asn Met Ile MetAla Glu Ser Pro Ser Leu Ile Thr 1 5 10 15 Ile Cys Leu Leu Gly Tyr LeuLeu Ser Ala Glu Cys Thr Val Phe Leu 20 25 30 Asp His Glu Asn Ala Asn LysIle Leu Asn Arg Pro Lys Arg Tyr Asn 35 40 45 Ser Gly Lys Leu Glu Glu PheVal Gln Gly Asn Leu Glu Arg Glu Cys 50 55 60 Met Glu Glu Lys Cys Ser PheGlu Glu Pro Arg Glu Val Phe Glu Asn 65 70 75 80 Thr Glu Lys Thr Thr GluPhe Trp Lys Gln Tyr Val Asp Gly Asp Gln 85 90 95 Cys Glu Ser Asn Pro CysLeu Asn Gly Gly Ser Cys Lys Asp Asp Ile 100 105 110 Asn Ser Tyr Glu CysTrp Cys Pro Phe Gly Phe Glu Gly Lys Asn Cys 115 120 125 Glu Leu Asp ValThr Cys Asn Ile Lys Asn Gly Arg Cys Glu Gln Phe 130 135 140 Cys Lys AsnSer Ala Asp Asn Lys Val Val Cys Ser Cys Thr Glu Gly 145 150 155 160 TyrArg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe 165 170 175Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala 180 185190 Glu Ala Val Phe Pro Asp Val Asp Tyr Val Asn Pro Thr Glu Ala Glu 195200 205 Thr Ile Leu Asp Asn Ile Thr Gln Gly Thr Gln Ser Phe Asn Asp Phe210 215 220 Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe ProTrp 225 230 235 240 Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys GlyGly Ser Ile 245 250 255 Val Asn Glu Lys Trp Ile Val Thr Ala Ala His CysVal Glu Thr Gly 260 265 270 Val Lys Ile Thr Val Val Ala Gly Glu His AsnIle Glu Glu Thr Glu 275 280 285 His Thr Glu Gln Lys Arg Asn Val Ile ArgAla Ile Ile Pro His His 290 295 300 Asn Tyr Asn Ala Ala Ile Asn Lys TyrAsn His Asp Ile Ala Leu Leu 305 310 315 320 Glu Leu Asp Glu Pro Leu ValLeu Asn Ser Tyr Val Thr Pro Ile Cys 325 330 335 Ile Ala Asp Lys Glu TyrThr Asn Ile Phe Leu Lys Phe Gly Ser Gly 340 345 350 Tyr Val Ser Gly TrpAla Arg Val Phe His Lys Gly Arg Ser Ala Leu 355 360 365 Val Leu Gln TyrLeu Arg Val Pro Leu Val Asp Arg Ala Thr Cys Leu 370 375 380 Arg Ser ThrLys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Phe 385 390 395 400 HisGlu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His 405 410 415Val Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser Trp 420 425430 Gly Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val 435440 445 Ser Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr 450 455460 11 603 DNA Homo sapiens 11 atggattact acagaaaata tgcagctatctttctggtca cattgtcggt gtttctgcat 60 gttctccatt ccgctcctga tgtgcaggattgcccagaat gcacgctaca ggaaaaccca 120 ttcttctccc agccgggtgc cccaatacttcagtgcatgg gctgctgctt ctctagagca 180 tatcccactc cactaaggtc caagaagacgatgttggtcc aaaagaacgt cacctcagag 240 tccacttgct gtgtagctaa atcatataacagggtcacag taatgggggg tttcaaagtg 300 gagaaccaca cggcgtgcca ctgcagtacttgttattatc acaaatctta aatgttttac 360 caagtgctgt cttgatgact gctgattttctggaatggaa aattaagttg tttagtgttt 420 atggctttgt gagataaaac tctccttttccttaccatac cactttgaca cgcttcaagg 480 atatactgca gctttactgc cttcctccttatcctacagt acaatcagca gtctagttct 540 tttcatttgg aatgaataca gcattaagcttgttccactg caaataaagc cttttaaatc 600 atc 603 12 116 PRT Homo sapiens 12Met Asp Tyr Tyr Arg Lys Tyr Ala Ala Ile Phe Leu Val Thr Leu Ser 1 5 1015 Val Phe Leu His Val Leu His Ser Ala Pro Asp Val Gln Asp Cys Pro 20 2530 Glu Cys Thr Leu Gln Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala Pro 35 4045 Ile Leu Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro 50 5560 Leu Arg Ser Lys Lys Thr Met Leu Val Gln Lys Asn Val Thr Ser Glu 65 7075 80 Ser Thr Cys Cys Val Ala Lys Ser Tyr Asn Arg Val Thr Val Met Gly 8590 95 Gly Phe Lys Val Glu Asn His Thr Ala Cys His Cys Ser Thr Cys Tyr100 105 110 Tyr His Lys Ser 115 13 390 DNA Homo sapiens 13 atgaagacactccagttttt cttccttttc tgttgctgga aagcaatctg ctgcaatagc 60 tgtgagctgaccaacatcac cattgcaata gagaaagaag aatgtcgttt ctgcataagc 120 atcaacaccacttggtgtgc tggctactgc tacaccaggg atctggtgta taaggaccca 180 gccaggcccaaaatccagaa aacatgtacc ttcaaggaac tggtatatga aacagtgaga 240 gtgcccggctgtgctcacca tgcagattcc ttgtatacat acccagtggc cacccagtgt 300 cactgtggcaagtgtgacag cgacagcact gattgtactg tgcgaggcct ggggcccagc 360 tactgctcctttggtgaaat gaaagaataa 390 14 129 PRT Homo sapiens 14 Met Lys Thr Leu GlnPhe Phe Phe Leu Phe Cys Cys Trp Lys Ala Ile 1 5 10 15 Cys Cys Asn SerCys Glu Leu Thr Asn Ile Thr Ile Ala Ile Glu Lys 20 25 30 Glu Glu Cys ArgPhe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala Gly 35 40 45 Tyr Cys Tyr ThrArg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Lys 50 55 60 Ile Gln Lys ThrCys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Arg 65 70 75 80 Val Pro GlyCys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val 85 90 95 Ala Thr GlnCys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys 100 105 110 Thr ValArg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu Met Lys 115 120 125 Glu15 1342 DNA Homo sapiens 15 cccggagccg gaccggggcc accgcgcccg ctctgctccgacaccgcgcc ccctggacag 60 ccgccctctc ctccaggccc gtggggctgg ccctgcaccgccgagcttcc cgggatgagg 120 gcccccggtg tggtcacccg gcgcgcccca ggtcgctgagggaccccggc caggcgcgga 180 gatgggggtg cacgaatgtc ctgcctggct gtggcttctcctgtccctgc tgtcgctccc 240 tctgggcctc ccagtcctgg gcgccccacc acgcctcatctgtgacagcc gagtcctgga 300 gaggtacctc ttggaggcca aggaggccga gaatatcacgacgggctgtg ctgaacactg 360 cagcttgaat gagaatatca ctgtcccaga caccaaagttaatttctatg cctggaagag 420 gatggaggtc gggcagcagg ccgtagaagt ctggcagggcctggccctgc tgtcggaagc 480 tgtcctgcgg ggccaggccc tgttggtcaa ctcttcccagccgtgggagc ccctgcagct 540 gcatgtggat aaagccgtca gtggccttcg cagcctcaccactctgcttc gggctctgcg 600 agcccagaag gaagccatct cccctccaga tgcggcctcagctgctccac tccgaacaat 660 cactgctgac actttccgca aactcttccg agtctactccaatttcctcc ggggaaagct 720 gaagctgtac acaggggagg cctgcaggac aggggacagatgaccaggtg tgtccacctg 780 ggcatatcca ccacctccct caccaacatt gcttgtgccacaccctcccc cgccactcct 840 gaaccccgtc gaggggctct cagctcagcg ccagcctgtcccatggacac tccagtgcca 900 gcaatgacat ctcaggggcc agaggaactg tccagagagcaactctgaga tctaaggatg 960 tcacagggcc aacttgaggg cccagagcag gaagcattcagagagcagct ttaaactcag 1020 ggacagagcc atgctgggaa gacgcctgag ctcactcggcaccctgcaaa atttgatgcc 1080 aggacacgct ttggaggcga tttacctgtt ttcgcacctaccatcaggga caggatgacc 1140 tggagaactt aggtggcaag ctgtgacttc tccaggtctcacgggcatgg gcactccctt 1200 ggtggcaaga gcccccttga caccggggtg gtgggaaccatgaagacagg atgggggctg 1260 gcctctggct ctcatggggt ccaagttttg tgtattcttcaacctcattg acaagaactg 1320 aaaccaccaa aaaaaaaaaa aa 1342 16 193 PRT Homosapiens 16 Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu SerLeu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro ProArg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu AlaLys Glu 35 40 45 Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser LeuAsn Glu 50 55 60 Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala TrpLys Arg 65 70 75 80 Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln GlyLeu Ala Leu 85 90 95 Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu ValAsn Ser Ser 100 105 110 Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp LysAla Val Ser Gly 115 120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala LeuArg Ala Gln Lys Glu 130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser AlaAla Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys LeuPhe Arg Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys Leu Lys Leu TyrThr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg 17 435 DNA Homosapiens 17 atgtggctgc agagcctgct gctcttgggc actgtggcct gcagcatctctgcacccgcc 60 cgctcgccca gccccagcac gcagccctgg gagcatgtga atgccatccaggaggcccgg 120 cgtctcctga acctgagtag agacactgct gctgagatga atgaaacagtagaagtcatc 180 tcagaaatgt ttgacctcca ggagccgacc tgcctacaga cccgcctggagctgtacaag 240 cagggcctgc ggggcagcct caccaagctc aagggcccct tgaccatgatggccagccac 300 tacaagcagc actgccctcc aaccccggaa acttcctgtg caacccagattatcaccttt 360 gaaagtttca aagagaacct gaaggacttt ctgcttgtca tcccctttgactgctgggag 420 ccagtccagg agtga 435 18 144 PRT Homo sapiens 18 Met TrpLeu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10 15 SerAla Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His 20 25 30 ValAsn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp 35 40 45 ThrAla Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe 50 55 60 AspLeu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys 65 70 75 80Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met 85 90 95Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser 100 105110 Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys 115120 125 Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu130 135 140 19 501 DNA Homo sapiens 19 atgaaatata caagttatat cttggcttttcagctctgca tcgttttggg ttctcttggc 60 tgttactgcc aggacccata tgtaaaagaagcagaaaacc ttaagaaata ttttaatgca 120 ggtcattcag atgtagcgga taatggaactcttttcttag gcattttgaa gaattggaaa 180 gaggagagtg acagaaaaat aatgcagagccaaattgtct ccttttactt caaacttttt 240 aaaaacttta aagatgacca gagcatccaaaagagtgtgg agaccatcaa ggaagacatg 300 aatgtcaagt ttttcaatag caacaaaaagaaacgagatg acttcgaaaa gctgactaat 360 tattcggtaa ctgacttgaa tgtccaacgcaaagcaatac atgaactcat ccaagtgatg 420 gctgaactgt cgccagcagc taaaacagggaagcgaaaaa ggagtcagat gctgtttcga 480 ggtcgaagag catcccagta a 501 20 166PRT Homo sapiens 20 Met Lys Tyr Thr Ser Tyr Ile Leu Ala Phe Gln Leu CysIle Val Leu 1 5 10 15 Gly Ser Leu Gly Cys Tyr Cys Gln Asp Pro Tyr ValLys Glu Ala Glu 20 25 30 Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser AspVal Ala Asp Asn 35 40 45 Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp LysGlu Glu Ser Asp 50 55 60 Arg Lys Ile Met Gln Ser Gln Ile Val Ser Phe TyrPhe Lys Leu Phe 65 70 75 80 Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln LysSer Val Glu Thr Ile 85 90 95 Lys Glu Asp Met Asn Val Lys Phe Phe Asn SerAsn Lys Lys Lys Arg 100 105 110 Asp Asp Phe Glu Lys Leu Thr Asn Tyr SerVal Thr Asp Leu Asn Val 115 120 125 Gln Arg Lys Ala Ile His Glu Leu IleGln Val Met Ala Glu Leu Ser 130 135 140 Pro Ala Ala Lys Thr Gly Lys ArgLys Arg Ser Gln Met Leu Phe Arg 145 150 155 160 Gly Arg Arg Ala Ser Gln165 21 1352 DNA Homo sapiens 21 ctgggacagt gaatcgacaa tgccgtcttctgtctcgtgg ggcatcctcc tgctggcagg 60 cctgtgctgc ctggtccctg tctccctggctgaggatccc cagggagatg ctgcccagaa 120 gacagataca tcccaccatg atcaggatcacccaaccttc aacaagatca cccccaacct 180 ggctgagttc gccttcagcc tataccgccagctggcacac cagtccaaca gcaccaatat 240 cttcttctcc ccagtgagca tcgctacagcctttgcaatg ctctccctgg ggaccaaggc 300 tgacactcac gatgaaatcc tggagggcctgaatttcaac ctcacggaga ttccggaggc 360 tcagatccat gaaggcttcc aggaactcctccgtaccctc aaccagccag acagccagct 420 ccagctgacc accggcaatg gcctgttcctcagcgagggc ctgaagctag tggataagtt 480 tttggaggat gttaaaaagt tgtaccactcagaagccttc actgtcaact tcggggacac 540 cgaagaggcc aagaaacaga tcaacgattacgtggagaag ggtactcaag ggaaaattgt 600 ggatttggtc aaggagcttg acagagacacagtttttgct ctggtgaatt acatcttctt 660 taaaggcaaa tgggagagac cctttgaagtcaaggacacc gaggaagagg acttccacgt 720 ggaccaggtg accaccgtga aggtgcctatgatgaagcgt ttaggcatgt ttaacatcca 780 gcactgtaag aagctgtcca gctgggtgctgctgatgaaa tacctgggca atgccaccgc 840 catcttcttc ctgcctgatg aggggaaactacagcacctg gaaaatgaac tcacccacga 900 tatcatcacc aagttcctgg aaaatgaagacagaaggtct gccagcttac atttacccaa 960 actgtccatt actggaacct atgatctgaagagcgtcctg ggtcaactgg gcatcactaa 1020 ggtcttcagc aatggggctg acctctccggggtcacagag gaggcacccc tgaagctctc 1080 caaggccgtg cataaggctg tgctgaccatcgacgagaaa gggactgaag ctgctggggc 1140 catgttttta gaggccatac ccatgtctatcccccccgag gtcaagttca acaaaccctt 1200 tgtcttctta atgattgaac aaaataccaagtctcccctc ttcatgggaa aagtggtgaa 1260 tcccacccaa aaataactgc ctctcgctcctcaacccctc ccctccatcc ctggccccct 1320 ccctggatga cattaaagaa gggttgagctgg 1352 22 418 PRT Homo sapiens 22 Met Pro Ser Ser Val Ser Trp Gly IleLeu Leu Leu Ala Gly Leu Cys 1 5 10 15 Cys Leu Val Pro Val Ser Leu AlaGlu Asp Pro Gln Gly Asp Ala Ala 20 25 30 Gln Lys Thr Asp Thr Ser His HisAsp Gln Asp His Pro Thr Phe Asn 35 40 45 Lys Ile Thr Pro Asn Leu Ala GluPhe Ala Phe Ser Leu Tyr Arg Gln 50 55 60 Leu Ala His Gln Ser Asn Ser ThrAsn Ile Phe Phe Ser Pro Val Ser 65 70 75 80 Ile Ala Thr Ala Phe Ala MetLeu Ser Leu Gly Thr Lys Ala Asp Thr 85 90 95 His Asp Glu Ile Leu Glu GlyLeu Asn Phe Asn Leu Thr Glu Ile Pro 100 105 110 Glu Ala Gln Ile His GluGly Phe Gln Glu Leu Leu Arg Thr Leu Asn 115 120 125 Gln Pro Asp Ser GlnLeu Gln Leu Thr Thr Gly Asn Gly Leu Phe Leu 130 135 140 Ser Glu Gly LeuLys Leu Val Asp Lys Phe Leu Glu Asp Val Lys Lys 145 150 155 160 Leu TyrHis Ser Glu Ala Phe Thr Val Asn Phe Gly Asp Thr Glu Glu 165 170 175 AlaLys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln Gly Lys 180 185 190Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val Phe Ala Leu 195 200205 Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro Phe Glu Val 210215 220 Lys Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val Thr Thr Val225 230 235 240 Lys Val Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile GlnHis Cys 245 250 255 Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr LeuGly Asn Ala 260 265 270 Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly Lys LeuGln His Leu Glu 275 280 285 Asn Glu Leu Thr His Asp Ile Ile Thr Lys PheLeu Glu Asn Glu Asp 290 295 300 Arg Arg Ser Ala Ser Leu His Leu Pro LysLeu Ser Ile Thr Gly Thr 305 310 315 320 Tyr Asp Leu Lys Ser Val Leu GlyGln Leu Gly Ile Thr Lys Val Phe 325 330 335 Ser Asn Gly Ala Asp Leu SerGly Val Thr Glu Glu Ala Pro Leu Lys 340 345 350 Leu Ser Lys Ala Val HisLys Ala Val Leu Thr Ile Asp Glu Lys Gly 355 360 365 Thr Glu Ala Ala GlyAla Met Phe Leu Glu Ala Ile Pro Met Ser Ile 370 375 380 Pro Pro Glu ValLys Phe Asn Lys Pro Phe Val Phe Leu Met Ile Glu 385 390 395 400 Gln AsnThr Lys Ser Pro Leu Phe Met Gly Lys Val Val Asn Pro Thr 405 410 415 GlnLys 23 2004 DNA Homo sapiens 23 gctaacctag tgcctatagc taaggcaggtacctgcatcc ttgtttttgt ttagtggatc 60 ctctatcctt cagagactct ggaacccctgtggtcttctc ttcatctaat gaccctgagg 120 ggatggagtt ttcaagtcct tccagagaggaatgtcccaa gcctttgagt agggtaagca 180 tcatggctgg cagcctcaca ggtttgcttctacttcaggc agtgtcgtgg gcatcaggtg 240 cccgcccctg catccctaaa agcttcggctacagctcggt ggtgtgtgtc tgcaatgcca 300 catactgtga ctcctttgac cccccgacctttcctgccct tggtaccttc agccgctatg 360 agagtacacg cagtgggcga cggatggagctgagtatggg gcccatccag gctaatcaca 420 cgggcacagg cctgctactg accctgcagccagaacagaa gttccagaaa gtgaagggat 480 ttggaggggc catgacagat gctgctgctctcaacatcct tgccctgtca ccccctgccc 540 aaaatttgct acttaaatcg tacttctctgaagaaggaat cggatataac atcatccggg 600 tacccatggc cagctgtgac ttctccatccgcacctacac ctatgcagac acccctgatg 660 atttccagtt gcacaacttc agcctcccagaggaagatac caagctcaag atacccctga 720 ttcaccgagc cctgcagttg gcccagcgtcccgtttcact ccttgccagc ccctggacat 780 cacccacttg gctcaagacc aatggagcggtgaatgggaa ggggtcactc aagggacagc 840 ccggagacat ctaccaccag acctgggccagatactttgt gaagttcctg gatgcctatg 900 ctgagcacaa gttacagttc tgggcagtgacagctgaaaa tgagccttct gctgggctgt 960 tgagtggata ccccttccag tgcctgggcttcacccctga acatcagcga gacttcattg 1020 cccgtgacct aggtcctacc ctcgccaacagtactcacca caatgtccgc ctactcatgc 1080 tggatgacca acgcttgctg ctgccccactgggcaaaggt ggtactgaca gacccagaag 1140 cagctaaata tgttcatggc attgctgtacattggtacct ggactttctg gctccagcca 1200 aagccaccct aggggagaca caccgcctgttccccaacac catgctcttt gcctcagagg 1260 cctgtgtggg ctccaagttc tgggagcagagtgtgcggct aggctcctgg gatcgaggga 1320 tgcagtacag ccacagcatc atcacgaacctcctgtacca tgtggtcggc tggaccgact 1380 ggaaccttgc cctgaacccc gaaggaggacccaattgggt gcgtaacttt gtcgacagtc 1440 ccatcattgt agacatcacc aaggacacgttttacaaaca gcccatgttc taccaccttg 1500 gccacttcag caagttcatt cctgagggctcccagagagt ggggctggtt gccagtcaga 1560 agaacgacct ggacgcagtg gcactgatgcatcccgatgg ctctgctgtt gtggtcgtgc 1620 taaaccgctc ctctaaggat gtgcctcttaccatcaagga tcctgctgtg ggcttcctgg 1680 agacaatctc acctggctac tccattcacacctacctgtg gcatcgccag tgatggagca 1740 gatactcaag gaggcactgg gctcagcctgggcattaaag ggacagagtc agctcacacg 1800 ctgtctgtga ctaaagaggg cacagcagggccagtgtgag cttacagcga cgtaagccca 1860 ggggcaatgg tttgggtgac tcactttcccctctaggtgg tgcccagggc tggaggcccc 1920 tagaaaaaga tcagtaagcc ccagtgtccccccagccccc atgcttatgt gaacatgcgc 1980 tgtgtgctgc ttgctttgga aact 2004 24536 PRT Homo sapiens 24 Met Glu Phe Ser Ser Pro Ser Arg Glu Glu Cys ProLys Pro Leu Ser 1 5 10 15 Arg Val Ser Ile Met Ala Gly Ser Leu Thr GlyLeu Leu Leu Leu Gln 20 25 30 Ala Val Ser Trp Ala Ser Gly Ala Arg Pro CysIle Pro Lys Ser Phe 35 40 45 Gly Tyr Ser Ser Val Val Cys Val Cys Asn AlaThr Tyr Cys Asp Ser 50 55 60 Phe Asp Pro Pro Thr Phe Pro Ala Leu Gly ThrPhe Ser Arg Tyr Glu 65 70 75 80 Ser Thr Arg Ser Gly Arg Arg Met Glu LeuSer Met Gly Pro Ile Gln 85 90 95 Ala Asn His Thr Gly Thr Gly Leu Leu LeuThr Leu Gln Pro Glu Gln 100 105 110 Lys Phe Gln Lys Val Lys Gly Phe GlyGly Ala Met Thr Asp Ala Ala 115 120 125 Ala Leu Asn Ile Leu Ala Leu SerPro Pro Ala Gln Asn Leu Leu Leu 130 135 140 Lys Ser Tyr Phe Ser Glu GluGly Ile Gly Tyr Asn Ile Ile Arg Val 145 150 155 160 Pro Met Ala Ser CysAsp Phe Ser Ile Arg Thr Tyr Thr Tyr Ala Asp 165 170 175 Thr Pro Asp AspPhe Gln Leu His Asn Phe Ser Leu Pro Glu Glu Asp 180 185 190 Thr Lys LeuLys Ile Pro Leu Ile His Arg Ala Leu Gln Leu Ala Gln 195 200 205 Arg ProVal Ser Leu Leu Ala Ser Pro Trp Thr Ser Pro Thr Trp Leu 210 215 220 LysThr Asn Gly Ala Val Asn Gly Lys Gly Ser Leu Lys Gly Gln Pro 225 230 235240 Gly Asp Ile Tyr His Gln Thr Trp Ala Arg Tyr Phe Val Lys Phe Leu 245250 255 Asp Ala Tyr Ala Glu His Lys Leu Gln Phe Trp Ala Val Thr Ala Glu260 265 270 Asn Glu Pro Ser Ala Gly Leu Leu Ser Gly Tyr Pro Phe Gln CysLeu 275 280 285 Gly Phe Thr Pro Glu His Gln Arg Asp Phe Ile Ala Arg AspLeu Gly 290 295 300 Pro Thr Leu Ala Asn Ser Thr His His Asn Val Arg LeuLeu Met Leu 305 310 315 320 Asp Asp Gln Arg Leu Leu Leu Pro His Trp AlaLys Val Val Leu Thr 325 330 335 Asp Pro Glu Ala Ala Lys Tyr Val His GlyIle Ala Val His Trp Tyr 340 345 350 Leu Asp Phe Leu Ala Pro Ala Lys AlaThr Leu Gly Glu Thr His Arg 355 360 365 Leu Phe Pro Asn Thr Met Leu PheAla Ser Glu Ala Cys Val Gly Ser 370 375 380 Lys Phe Trp Glu Gln Ser ValArg Leu Gly Ser Trp Asp Arg Gly Met 385 390 395 400 Gln Tyr Ser His SerIle Ile Thr Asn Leu Leu Tyr His Val Val Gly 405 410 415 Trp Thr Asp TrpAsn Leu Ala Leu Asn Pro Glu Gly Gly Pro Asn Trp 420 425 430 Val Arg AsnPhe Val Asp Ser Pro Ile Ile Val Asp Ile Thr Lys Asp 435 440 445 Thr PheTyr Lys Gln Pro Met Phe Tyr His Leu Gly His Phe Ser Lys 450 455 460 PheIle Pro Glu Gly Ser Gln Arg Val Gly Leu Val Ala Ser Gln Lys 465 470 475480 Asn Asp Leu Asp Ala Val Ala Leu Met His Pro Asp Gly Ser Ala Val 485490 495 Val Val Val Leu Asn Arg Ser Ser Lys Asp Val Pro Leu Thr Ile Lys500 505 510 Asp Pro Ala Val Gly Phe Leu Glu Thr Ile Ser Pro Gly Tyr SerIle 515 520 525 His Thr Tyr Leu Trp His Arg Gln 530 535 25 1726 DNA Homosapiens 25 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagcagtcttcgtt 60 tcgcccagcc aggaaatcca tgcccgattc agaagaggag ccagatcttaccaagtgatc 120 tgcagagatg aaaaaacgca gatgatatac cagcaacatc agtcatggctgcgccctgtg 180 ctcagaagca accgggtgga atattgctgg tgcaacagtg gcagggcacagtgccactca 240 gtgcctgtca aaagttgcag cgagccaagg tgtttcaacg ggggcacctgccagcaggcc 300 ctgtacttct cagatttcgt gtgccagtgc cccgaaggat ttgctgggaagtgctgtgaa 360 atagatacca gggccacgtg ctacgaggac cagggcatca gctacaggggcacgtggagc 420 acagcggaga gtggcgccga gtgcaccaac tggaacagca gcgcgttggcccagaagccc 480 tacagcgggc ggaggccaga cgccatcagg ctgggcctgg ggaaccacaactactgcaga 540 aacccagatc gagactcaaa gccctggtgc tacgtcttta aggcggggaagtacagctca 600 gagttctgca gcacccctgc ctgctctgag ggaaacagtg actgctactttgggaatggg 660 tcagcctacc gtggcacgca cagcctcacc gagtcgggtg cctcctgcctcccgtggaat 720 tccatgatcc tgataggcaa ggtttacaca gcacagaacc ccagtgcccaggcactgggc 780 ctgggcaaac ataattactg ccggaatcct gatggggatg ccaagccctggtgccacgtg 840 ctgaagaacc gcaggctgac gtgggagtac tgtgatgtgc cctcctgctccacctgcggc 900 ctgagacagt acagccagcc tcagtttcgc atcaaaggag ggctcttcgccgacatcgcc 960 tcccacccct ggcaggctgc catctttgcc aagcacagga ggtcgccgggagagcggttc 1020 ctgtgcgggg gcatactcat cagctcctgc tggattctct ctgccgcccactgcttccag 1080 gagaggtttc cgccccacca cctgacggtg atcttgggca gaacataccgggtggtccct 1140 ggcgaggagg agcagaaatt tgaagtcgaa aaatacattg tccataaggaattcgatgat 1200 gacacttacg acaatgacat tgcgctgctg cagctgaaat cggattcgtcccgctgtgcc 1260 caggagagca gcgtggtccg cactgtgtgc cttcccccgg cggacctgcagctgccggac 1320 tggacggagt gtgagctctc cggctacggc aagcatgagg ccttgtctcctttctattcg 1380 gagcggctga aggaggctca tgtcagactg tacccatcca gccgctgcacatcacaacat 1440 ttacttaaca gaacagtcac cgacaacatg ctgtgtgctg gagacactcggagcggcggg 1500 ccccaggcaa acttgcacga cgcctgccag ggcgattcgg gaggccccctggtgtgtctg 1560 aacgatggcc gcatgacttt ggtgggcatc atcagctggg gcctgggctgtggacagaag 1620 gatgtcccgg gtgtgtacac caaggttacc aactacctag actggattcgtgacaacatg 1680 cgaccgtgac caggaacacc cgactcctca aaagcaaatg agatcc 172626 562 PRT Homo sapiens 26 Met Asp Ala Met Lys Arg Gly Leu Cys Cys ValLeu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Ser Gln Glu IleHis Ala Arg Phe Arg Arg 20 25 30 Gly Ala Arg Ser Tyr Gln Val Ile Cys ArgAsp Glu Lys Thr Gln Met 35 40 45 Ile Tyr Gln Gln His Gln Ser Trp Leu ArgPro Val Leu Arg Ser Asn 50 55 60 Arg Val Glu Tyr Cys Trp Cys Asn Ser GlyArg Ala Gln Cys His Ser 65 70 75 80 Val Pro Val Lys Ser Cys Ser Glu ProArg Cys Phe Asn Gly Gly Thr 85 90 95 Cys Gln Gln Ala Leu Tyr Phe Ser AspPhe Val Cys Gln Cys Pro Glu 100 105 110 Gly Phe Ala Gly Lys Cys Cys GluIle Asp Thr Arg Ala Thr Cys Tyr 115 120 125 Glu Asp Gln Gly Ile Ser TyrArg Gly Thr Trp Ser Thr Ala Glu Ser 130 135 140 Gly Ala Glu Cys Thr AsnTrp Asn Ser Ser Ala Leu Ala Gln Lys Pro 145 150 155 160 Tyr Ser Gly ArgArg Pro Asp Ala Ile Arg Leu Gly Leu Gly Asn His 165 170 175 Asn Tyr CysArg Asn Pro Asp Arg Asp Ser Lys Pro Trp Cys Tyr Val 180 185 190 Phe LysAla Gly Lys Tyr Ser Ser Glu Phe Cys Ser Thr Pro Ala Cys 195 200 205 SerGlu Gly Asn Ser Asp Cys Tyr Phe Gly Asn Gly Ser Ala Tyr Arg 210 215 220Gly Thr His Ser Leu Thr Glu Ser Gly Ala Ser Cys Leu Pro Trp Asn 225 230235 240 Ser Met Ile Leu Ile Gly Lys Val Tyr Thr Ala Gln Asn Pro Ser Ala245 250 255 Gln Ala Leu Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro AspGly 260 265 270 Asp Ala Lys Pro Trp Cys His Val Leu Lys Asn Arg Arg LeuThr Trp 275 280 285 Glu Tyr Cys Asp Val Pro Ser Cys Ser Thr Cys Gly LeuArg Gln Tyr 290 295 300 Ser Gln Pro Gln Phe Arg Ile Lys Gly Gly Leu PheAla Asp Ile Ala 305 310 315 320 Ser His Pro Trp Gln Ala Ala Ile Phe AlaLys His Arg Arg Ser Pro 325 330 335 Gly Glu Arg Phe Leu Cys Gly Gly IleLeu Ile Ser Ser Cys Trp Ile 340 345 350 Leu Ser Ala Ala His Cys Phe GlnGlu Arg Phe Pro Pro His His Leu 355 360 365 Thr Val Ile Leu Gly Arg ThrTyr Arg Val Val Pro Gly Glu Glu Glu 370 375 380 Gln Lys Phe Glu Val GluLys Tyr Ile Val His Lys Glu Phe Asp Asp 385 390 395 400 Asp Thr Tyr AspAsn Asp Ile Ala Leu Leu Gln Leu Lys Ser Asp Ser 405 410 415 Ser Arg CysAla Gln Glu Ser Ser Val Val Arg Thr Val Cys Leu Pro 420 425 430 Pro AlaAsp Leu Gln Leu Pro Asp Trp Thr Glu Cys Glu Leu Ser Gly 435 440 445 TyrGly Lys His Glu Ala Leu Ser Pro Phe Tyr Ser Glu Arg Leu Lys 450 455 460Glu Ala His Val Arg Leu Tyr Pro Ser Ser Arg Cys Thr Ser Gln His 465 470475 480 Leu Leu Asn Arg Thr Val Thr Asp Asn Met Leu Cys Ala Gly Asp Thr485 490 495 Arg Ser Gly Gly Pro Gln Ala Asn Leu His Asp Ala Cys Gln GlyAsp 500 505 510 Ser Gly Gly Pro Leu Val Cys Leu Asn Asp Gly Arg Met ThrLeu Val 515 520 525 Gly Ile Ile Ser Trp Gly Leu Gly Cys Gly Gln Lys AspVal Pro Gly 530 535 540 Val Tyr Thr Lys Val Thr Asn Tyr Leu Asp Trp IleArg Asp Asn Met 545 550 555 560 Arg Pro 27 825 DNA Homo sapiens 27atcactctct ttaatcacta ctcacattaa cctcaactcc tgccacaatg tacaggatgc 60aactcctgtc ttgcattgca ctaattcttg cacttgtcac aaacagtgca cctacttcaa 120gttcgacaaa gaaaacaaag aaaacacagc tacaactgga gcatttactg ctggatttac 180agatgatttt gaatggaatt aataattaca agaatcccaa actcaccagg atgctcacat 240ttaagtttta catgcccaag aaggccacag aactgaaaca gcttcagtgt ctagaagaag 300aactcaaacc tctggaggaa gtgctgaatt tagctcaaag caaaaacttt cacttaagac 360ccagggactt aatcagcaat atcaacgtaa tagttctgga actaaaggga tctgaaacaa 420cattcatgtg tgaatatgca gatgagacag caaccattgt agaatttctg aacagatgga 480ttaccttttg tcaaagcatc atctcaacac taacttgata attaagtgct tcccacttaa 540aacatatcag gccttctatt tatttattta aatatttaaa ttttatattt attgttgaat 600gtatggttgc tacctattgt aactattatt cttaatctta aaactataaa tatggatctt 660ttatgattct ttttgtaagc cctaggggct ctaaaatggt ttaccttatt tatcccaaaa 720atatttatta ttatgttgaa tgttaaatat agtatctatg tagattggtt agtaaaacta 780tttaataaat ttgataaata taaaaaaaaa aaacaaaaaa aaaaa 825 28 156 PRT Homosapiens 28 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ile Leu AlaLeu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys ThrLys Lys 20 25 30 Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln MetIle Leu 35 40 45 Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg MetLeu Thr 50 55 60 Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys GlnLeu Gln 65 70 75 80 Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val LeuAsn Leu Ala 85 90 95 Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu IleSer Asn Ile 100 105 110 Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu ThrThr Phe Met Cys 115 120 125 Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val GluPhe Leu Asn Arg Trp 130 135 140 Ile Thr Phe Cys Gln Ser Ile Ile Ser ThrLeu Thr 145 150 155 29 7931 DNA Homo sapiens 29 atgcaaatag agctctccacctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60 accagaagat actacctgggtgcagtggaa ctgtcatggg actatatgca aagtgatctc 120 ggtgagctgc ctgtggacgcaagatttcct cctagagtgc caaaatcttt tccattcaac 180 acctcagtcg tgtacaaaaagactctgttt gtagaattca cggatcacct tttcaacatc 240 gctaagccaa ggccaccctggatgggtctg ctaggtccta ccatccaggc tgaggtttat 300 gatacagtgg tcattacacttaagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360 ggtgtatcct actggaaagcttctgaggga gctgaatatg atgatcagac cagtcaaagg 420 gagaaagaag atgataaagtcttccctggt ggaagccata catatgtctg gcaggtcctg 480 aaagagaatg gtccaatggcctctgaccca ctgtgcctta cctactcata tctttctcat 540 gtggacctgg taaaagacttgaattcaggc ctcattggag ccctactagt atgtagagaa 600 gggagtctgg ccaaggaaaagacacagacc ttgcacaaat ttatactact ttttgctgta 660 tttgatgaag ggaaaagttggcactcagaa acaaagaact ccttgatgca ggatagggat 720 gctgcatctg ctcgggcctggcctaaaatg cacacagtca atggttatgt aaacaggtct 780 ctgccaggtc tgattggatgccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840 accactcctg aagtgcactcaatattcctc gaaggtcaca catttcttgt gaggaaccat 900 cgccaggcgt ccttggaaatctcgccaata actttcctta ctgctcaaac actcttgatg 960 gaccttggac agtttctactgttttgtcat atctcttccc accaacatga tggcatggaa 1020 gcttatgtca aagtagacagctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080 gaagcggaag actatgatgatgatcttact gattctgaaa tggatgtggt caggtttgat 1140 gatgacaact ctccttcctttatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200 tgggtacatt acattgctgctgaagaggag gactgggact atgctccctt agtcctcgcc 1260 cccgatgaca gaagttataaaagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320 aagtacaaaa aagtccgatttatggcatac acagatgaaa cctttaagac tcgtgaagct 1380 attcagcatg aatcaggaatcttgggacct ttactttatg gggaagttgg agacacactg 1440 ttgattatat ttaagaatcaagcaagcaga ccatataaca tctaccctca cggaatcact 1500 gatgtccgtc ctttgtattcaaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560 ccaattctgc caggagaaatattcaaatat aaatggacag tgactgtaga agatgggcca 1620 actaaatcag atcctcggtgcctgacccgc tattactcta gtttcgttaa tatggagaga 1680 gatctagctt caggactcattggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740 agaggaaacc agataatgtcagacaagagg aatgtcatcc tgttttctgt atttgatgag 1800 aaccgaagct ggtacctcacagagaatata caacgctttc tccccaatcc agctggagtg 1860 cagcttgagg atccagagttccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920 tttgatagtt tgcagttgtcagtttgtttg catgaggtgg catactggta cattctaagc 1980 attggagcac agactgacttcctttctgtc ttcttctctg gatatacctt caaacacaaa 2040 atggtctatg aagacacactcaccctattc ccattctcag gagaaactgt cttcatgtcg 2100 atggaaaacc caggtctatggattctgggg tgccacaact cagactttcg gaacagaggc 2160 atgaccgcct tactgaaggtttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220 agttatgaag atatttcagcatacttgctg agtaaaaaca atgccattga accaagaagc 2280 ttctcccaga attcaagacaccgtagcact aggcaaaagc aatttaatgc caccacaatt 2340 ccagaaaatg acatagagaagactgaccct tggtttgcac acagaacacc tatgcctaaa 2400 atacaaaatg tctcctctagtgatttgttg atgctcttgc gacagagtcc tactccacat 2460 gggctatcct tatctgatctccaagaagcc aaatatgaga ctttttctga tgatccatca 2520 cctggagcaa tagacagtaataacagcctg tctgaaatga cacacttcag gccacagctc 2580 catcacagtg gggacatggtatttacccct gagtcaggcc tccaattaag attaaatgag 2640 aaactgggga caactgcagcaacagagttg aagaaacttg atttcaaagt ttctagtaca 2700 tcaaataatc tgatttcaacaattccatca gacaatttgg cagcaggtac tgataataca 2760 agttccttag gacccccaagtatgccagtt cattatgata gtcaattaga taccactcta 2820 tttggcaaaa agtcatctccccttactgag tctggtggac ctctgagctt gagtgaagaa 2880 aataatgatt caaagttgttagaatcaggt ttaatgaata gccaagaaag ttcatgggga 2940 aaaaatgtat cgtcaacagagagtggtagg ttatttaaag ggaaaagagc tcatggacct 3000 gctttgttga ctaaagataatgccttattc aaagttagca tctctttgtt aaagacaaac 3060 aaaacttcca ataattcagcaactaataga aagactcaca ttgatggccc atcattatta 3120 attgagaata gtccatcagtctggcaaaat atattagaaa gtgacactga gtttaaaaaa 3180 gtgacacctt tgattcatgacagaatgctt atggacaaaa atgctacagc tttgaggcta 3240 aatcatatgt caaataaaactacttcatca aaaaacatgg aaatggtcca acagaaaaaa 3300 gagggcccca ttccaccagatgcacaaaat ccagatatgt cgttctttaa gatgctattc 3360 ttgccagaat cagcaaggtggatacaaagg actcatggaa agaactctct gaactctggg 3420 caaggcccca gtccaaagcaattagtatcc ttaggaccag aaaaatctgt ggaaggtcag 3480 aatttcttgt ctgagaaaaacaaagtggta gtaggaaagg gtgaatttac aaaggacgta 3540 ggactcaaag agatggtttttccaagcagc agaaacctat ttcttactaa cttggataat 3600 ttacatgaaa ataatacacacaatcaagaa aaaaaaattc aggaagaaat agaaaagaag 3660 gaaacattaa tccaagagaatgtagttttg cctcagatac atacagtgac tggcactaag 3720 aatttcatga agaaccttttcttactgagc actaggcaaa atgtagaagg ttcatatgac 3780 ggggcatatg ctccagtacttcaagatttt aggtcattaa atgattcaac aaatagaaca 3840 aagaaacaca cagctcatttctcaaaaaaa ggggaggaag aaaacttgga aggcttggga 3900 aatcaaacca agcaaattgtagagaaatat gcatgcacca caaggatatc tcctaataca 3960 agccagcaga attttgtcacgcaacgtagt aagagagctt tgaaacaatt cagactccca 4020 ctagaagaaa cagaacttgaaaaaaggata attgtggatg acacctcaac ccagtggtcc 4080 aaaaacatga aacatttgaccccgagcacc ctcacacaga tagactacaa tgagaaggag 4140 aaaggggcca ttactcagtctcccttatca gattgcctta cgaggagtca tagcatccct 4200 caagcaaata gatctccattacccattgca aaggtatcat catttccatc tattagacct 4260 atatatctga ccagggtcctattccaagac aactcttctc atcttccagc agcatcttat 4320 agaaagaaag attctggggtccaagaaagc agtcatttct tacaaggagc caaaaaaaat 4380 aacctttctt tagccattctaaccttggag atgactggtg atcaaagaga ggttggctcc 4440 ctggggacaa gtgccacaaattcagtcaca tacaagaaag ttgagaacac tgttctcccg 4500 aaaccagact tgcccaaaacatctggcaaa gttgaattgc ttccaaaagt tcacatttat 4560 cagaaggacc tattccctacggaaactagc aatgggtctc ctggccatct ggatctcgtg 4620 gaagggagcc ttcttcagggaacagaggga gcgattaagt ggaatgaagc aaacagacct 4680 ggaaaagttc cctttctgagagtagcaaca gaaagctctg caaagactcc ctccaagcta 4740 ttggatcctc ttgcttgggataaccactat ggtactcaga taccaaaaga agagtggaaa 4800 tcccaagaga agtcaccagaaaaaacagct tttaagaaaa aggataccat tttgtccctg 4860 aacgcttgtg aaagcaatcatgcaatagca gcaataaatg agggacaaaa taagcccgaa 4920 atagaagtca cctgggcaaagcaaggtagg actgaaaggc tgtgctctca aaacccacca 4980 gtcttgaaac gccatcaacgggaaataact cgtactactc ttcagtcaga tcaagaggaa 5040 attgactatg atgataccatatcagttgaa atgaagaagg aagattttga catttatgat 5100 gaggatgaaa atcagagcccccgcagcttt caaaagaaaa cacgacacta ttttattgct 5160 gcagtggaga ggctctgggattatgggatg agtagctccc cacatgttct aagaaacagg 5220 gctcagagtg gcagtgtccctcagttcaag aaagttgttt tccaggaatt tactgatggc 5280 tcctttactc agcccttataccgtggagaa ctaaatgaac atttgggact cctggggcca 5340 tatataagag cagaagttgaagataatatc atggtaactt tcagaaatca ggcctctcgt 5400 ccctattcct tctattctagccttatttct tatgaggaag atcagaggca aggagcagaa 5460 cctagaaaaa actttgtcaagcctaatgaa accaaaactt acttttggaa agtgcaacat 5520 catatggcac ccactaaagatgagtttgac tgcaaagcct gggcttattt ctctgatgtt 5580 gacctggaaa aagatgtgcactcaggcctg attggacccc ttctggtctg ccacactaac 5640 acactgaacc ctgctcatgggagacaagtg acagtacagg aatttgctct gtttttcacc 5700 atctttgatg agaccaaaagctggtacttc actgaaaata tggaaagaaa ctgcagggct 5760 ccctgcaata tccagatggaagatcccact tttaaagaga attatcgctt ccatgcaatc 5820 aatggctaca taatggatacactacctggc ttagtaatgg ctcaggatca aaggattcga 5880 tggtatctgc tcagcatgggcagcaatgaa aacatccatt ctattcattt cagtggacat 5940 gtgttcactg tacgaaaaaaagaggagtat aaaatggcac tgtacaatct ctatccaggt 6000 gtttttgaga cagtggaaatgttaccatcc aaagctggaa tttggcgggt ggaatgcctt 6060 attggcgagc atctacatgctgggatgagc acactttttc tggtgtacag caataagtgt 6120 cagactcccc tgggaatggcttctggacac attagagatt ttcagattac agcttcagga 6180 caatatggac agtgggccccaaagctggcc agacttcatt attccggatc aatcaatgcc 6240 tggagcacca aggagcccttttcttggatc aaggtggatc tgttggcacc aatgattatt 6300 cacggcatca agacccagggtgcccgtcag aagttctcca gcctctacat ctctcagttt 6360 atcatcatgt atagtcttgatgggaagaag tggcagactt atcgaggaaa ttccactgga 6420 accttaatgg tcttctttggcaatgtggat tcatctggga taaaacacaa tatttttaac 6480 cctccaatta ttgctcgatacatccgtttg cacccaactc attatagcat tcgcagcact 6540 cttcgcatgg agttgatgggctgtgattta aatagttgca gcatgccatt gggaatggag 6600 agtaaagcaa tatcagatgcacagattact gcttcatcct actttaccaa tatgtttgcc 6660 acctggtctc cttcaaaagctcgacttcac ctccaaggga ggagtaatgc ctggagacct 6720 caggtgaata atccaaaagagtggctgcaa gtggacttcc agaagacaat gaaagtcaca 6780 ggagtaacta ctcagggagtaaaatctctg cttaccagca tgtatgtgaa ggagttcctc 6840 atctccagca gtcaagatggccatcagtgg actctctttt ttcagaatgg caaagtaaag 6900 gtttttcagg gaaatcaagactccttcaca cctgtggtga actctctaga cccaccgtta 6960 ctgactcgct accttcgaattcacccccag agttgggtgc accagattgc cctgaggatg 7020 gaggttctgg gctgcgaggcacaggacctc tactgagggt ggccactgca gcacctgcca 7080 ctgccgtcac ctctccctcctcagctccag ggcagtgtcc ctccctggct tgccttctac 7140 ctttgtgcta aatcctagcagacactgcct tgaagcctcc tgaattaact atcatcagtc 7200 ctgcatttct ttggtggggggccaggaggg tgcatccaat ttaacttaac tcttacctat 7260 tttctgcagc tgctcccagattactccttc cttccaatat aactaggcaa aaagaagtga 7320 ggagaaacct gcatgaaagcattcttccct gaaaagttag gcctctcaga gtcaccactt 7380 cctctgttgt agaaaaactatgtgatgaaa ctttgaaaaa gatatttatg atgttaacat 7440 ttcaggttaa gcctcatacgtttaaaataa aactctcagt tgtttattat cctgatcaag 7500 catggaacaa agcatgtttcaggatcagat caatacaatc ttggagtcaa aaggcaaatc 7560 atttggacaa tctgcaaaatggagagaata caataactac tacagtaaag tctgtttctg 7620 cttccttaca catagatataattatgttat ttagtcatta tgaggggcac attcttatct 7680 ccaaaactag cattcttaaactgagaatta tagatggggt tcaagaatcc ctaagtcccc 7740 tgaaattata taaggcattctgtataaatg caaatgtgca tttttctgac gagtgtccat 7800 agatataaag ccatttggtcttaattctga ccaataaaaa aataagtcag gaggatgcaa 7860 ttgttgaaag ctttgaaataaaataacaat gtcttcttga aatttgtgat ggccaagaaa 7920 gaaaatgatg a 7931 302351 PRT Homo sapiens 30 Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu CysLeu Leu Arg Phe 1 5 10 15 Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu GlyAla Val Glu Leu Ser 20 25 30 Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu LeuPro Val Asp Ala Arg 35 40 45 Phe Pro Pro Arg Val Pro Lys Ser Phe Pro PheAsn Thr Ser Val Val 50 55 60 Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr AspHis Leu Phe Asn Ile 65 70 75 80 Ala Lys Pro Arg Pro Pro Trp Met Gly LeuLeu Gly Pro Thr Ile Gln 85 90 95 Ala Glu Val Tyr Asp Thr Val Val Ile ThrLeu Lys Asn Met Ala Ser 100 105 110 His Pro Val Ser Leu His Ala Val GlyVal Ser Tyr Trp Lys Ala Ser 115 120 125 Glu Gly Ala Glu Tyr Asp Asp GlnThr Ser Gln Arg Glu Lys Glu Asp 130 135 140 Asp Lys Val Phe Pro Gly GlySer His Thr Tyr Val Trp Gln Val Leu 145 150 155 160 Lys Glu Asn Gly ProMet Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser 165 170 175 Tyr Leu Ser HisVal Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile 180 185 190 Gly Ala LeuLeu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr 195 200 205 Gln ThrLeu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly 210 215 220 LysSer Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp 225 230 235240 Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr 245250 255 Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val260 265 270 Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His SerIle 275 280 285 Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg GlnAla Ser 290 295 300 Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln ThrLeu Leu Met 305 310 315 320 Asp Leu Gly Gln Phe Leu Leu Phe Cys His IleSer Ser His Gln His 325 330 335 Asp Gly Met Glu Ala Tyr Val Lys Val AspSer Cys Pro Glu Glu Pro 340 345 350 Gln Leu Arg Met Lys Asn Asn Glu GluAla Glu Asp Tyr Asp Asp Asp 355 360 365 Leu Thr Asp Ser Glu Met Asp ValVal Arg Phe Asp Asp Asp Asn Ser 370 375 380 Pro Ser Phe Ile Gln Ile ArgSer Val Ala Lys Lys His Pro Lys Thr 385 390 395 400 Trp Val His Tyr IleAla Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro 405 410 415 Leu Val Leu AlaPro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn 420 425 430 Asn Gly ProGln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met 435 440 445 Ala TyrThr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460 SerGly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475480 Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485490 495 His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys500 505 510 Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu IlePhe 515 520 525 Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr LysSer Asp 530 535 540 Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val AsnMet Glu Arg 545 550 555 560 Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu LeuIle Cys Tyr Lys Glu 565 570 575 Ser Val Asp Gln Arg Gly Asn Gln Ile MetSer Asp Lys Arg Asn Val 580 585 590 Ile Leu Phe Ser Val Phe Asp Glu AsnArg Ser Trp Tyr Leu Thr Glu 595 600 605 Asn Ile Gln Arg Phe Leu Pro AsnPro Ala Gly Val Gln Leu Glu Asp 610 615 620 Pro Glu Phe Gln Ala Ser AsnIle Met His Ser Ile Asn Gly Tyr Val 625 630 635 640 Phe Asp Ser Leu GlnLeu Ser Val Cys Leu His Glu Val Ala Tyr Trp 645 650 655 Tyr Ile Leu SerIle Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe 660 665 670 Ser Gly TyrThr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr 675 680 685 Leu PhePro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700 GlyLeu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly 705 710 715720 Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725730 735 Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys740 745 750 Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg HisArg 755 760 765 Ser Thr Arg Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro GluAsn Asp 770 775 780 Ile Glu Lys Thr Asp Pro Trp Phe Ala His Arg Thr ProMet Pro Lys 785 790 795 800 Ile Gln Asn Val Ser Ser Ser Asp Leu Leu MetLeu Leu Arg Gln Ser 805 810 815 Pro Thr Pro His Gly Leu Ser Leu Ser AspLeu Gln Glu Ala Lys Tyr 820 825 830 Glu Thr Phe Ser Asp Asp Pro Ser ProGly Ala Ile Asp Ser Asn Asn 835 840 845 Ser Leu Ser Glu Met Thr His PheArg Pro Gln Leu His His Ser Gly 850 855 860 Asp Met Val Phe Thr Pro GluSer Gly Leu Gln Leu Arg Leu Asn Glu 865 870 875 880 Lys Leu Gly Thr ThrAla Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys 885 890 895 Val Ser Ser ThrSer Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn 900 905 910 Leu Ala AlaGly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met 915 920 925 Pro ValHis Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys 930 935 940 SerSer Pro Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu 945 950 955960 Asn Asn Asp Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu 965970 975 Ser Ser Trp Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe980 985 990 Lys Gly Lys Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp AsnAla 995 1000 1005 Leu Phe Lys Val Ser Ile Ser Leu Leu Lys Thr Asn LysThr Ser 1010 1015 1020 Asn Asn Ser Ala Thr Asn Arg Lys Thr His Ile AspGly Pro Ser 1025 1030 1035 Leu Leu Ile Glu Asn Ser Pro Ser Val Trp GlnAsn Ile Leu Glu 1040 1045 1050 Ser Asp Thr Glu Phe Lys Lys Val Thr ProLeu Ile His Asp Arg 1055 1060 1065 Met Leu Met Asp Lys Asn Ala Thr AlaLeu Arg Leu Asn His Met 1070 1075 1080 Ser Asn Lys Thr Thr Ser Ser LysAsn Met Glu Met Val Gln Gln 1085 1090 1095 Lys Lys Glu Gly Pro Ile ProPro Asp Ala Gln Asn Pro Asp Met 1100 1105 1110 Ser Phe Phe Lys Met LeuPhe Leu Pro Glu Ser Ala Arg Trp Ile 1115 1120 1125 Gln Arg Thr His GlyLys Asn Ser Leu Asn Ser Gly Gln Gly Pro 1130 1135 1140 Ser Pro Lys GlnLeu Val Ser Leu Gly Pro Glu Lys Ser Val Glu 1145 1150 1155 Gly Gln AsnPhe Leu Ser Glu Lys Asn Lys Val Val Val Gly Lys 1160 1165 1170 Gly GluPhe Thr Lys Asp Val Gly Leu Lys Glu Met Val Phe Pro 1175 1180 1185 SerSer Arg Asn Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu 1190 1195 1200Asn Asn Thr His Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu 1205 12101215 Lys Lys Glu Thr Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile 12201225 1230 His Thr Val Thr Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu1235 1240 1245 Leu Ser Thr Arg Gln Asn Val Glu Gly Ser Tyr Asp Gly AlaTyr 1250 1255 1260 Ala Pro Val Leu Gln Asp Phe Arg Ser Leu Asn Asp SerThr Asn 1265 1270 1275 Arg Thr Lys Lys His Thr Ala His Phe Ser Lys LysGly Glu Glu 1280 1285 1290 Glu Asn Leu Glu Gly Leu Gly Asn Gln Thr LysGln Ile Val Glu 1295 1300 1305 Lys Tyr Ala Cys Thr Thr Arg Ile Ser ProAsn Thr Ser Gln Gln 1310 1315 1320 Asn Phe Val Thr Gln Arg Ser Lys ArgAla Leu Lys Gln Phe Arg 1325 1330 1335 Leu Pro Leu Glu Glu Thr Glu LeuGlu Lys Arg Ile Ile Val Asp 1340 1345 1350 Asp Thr Ser Thr Gln Trp SerLys Asn Met Lys His Leu Thr Pro 1355 1360 1365 Ser Thr Leu Thr Gln IleAsp Tyr Asn Glu Lys Glu Lys Gly Ala 1370 1375 1380 Ile Thr Gln Ser ProLeu Ser Asp Cys Leu Thr Arg Ser His Ser 1385 1390 1395 Ile Pro Gln AlaAsn Arg Ser Pro Leu Pro Ile Ala Lys Val Ser 1400 1405 1410 Ser Phe ProSer Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu Phe 1415 1420 1425 Gln AspAsn Ser Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys 1430 1435 1440 AspSer Gly Val Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys 1445 1450 1455Lys Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly 1460 14651470 Asp Gln Arg Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser 14751480 1485 Val Thr Tyr Lys Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp1490 1495 1500 Leu Pro Lys Thr Ser Gly Lys Val Glu Leu Leu Pro Lys ValHis 1505 1510 1515 Ile Tyr Gln Lys Asp Leu Phe Pro Thr Glu Thr Ser AsnGly Ser 1520 1525 1530 Pro Gly His Leu Asp Leu Val Glu Gly Ser Leu LeuGln Gly Thr 1535 1540 1545 Glu Gly Ala Ile Lys Trp Asn Glu Ala Asn ArgPro Gly Lys Val 1550 1555 1560 Pro Phe Leu Arg Val Ala Thr Glu Ser SerAla Lys Thr Pro Ser 1565 1570 1575 Lys Leu Leu Asp Pro Leu Ala Trp AspAsn His Tyr Gly Thr Gln 1580 1585 1590 Ile Pro Lys Glu Glu Trp Lys SerGln Glu Lys Ser Pro Glu Lys 1595 1600 1605 Thr Ala Phe Lys Lys Lys AspThr Ile Leu Ser Leu Asn Ala Cys 1610 1615 1620 Glu Ser Asn His Ala IleAla Ala Ile Asn Glu Gly Gln Asn Lys 1625 1630 1635 Pro Glu Ile Glu ValThr Trp Ala Lys Gln Gly Arg Thr Glu Arg 1640 1645 1650 Leu Cys Ser GlnAsn Pro Pro Val Leu Lys Arg His Gln Arg Glu 1655 1660 1665 Ile Thr ArgThr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr 1670 1675 1680 Asp AspThr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile 1685 1690 1695 TyrAsp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys 1700 1705 1710Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr 1715 17201725 Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser 17301735 1740 Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr1745 1750 1755 Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu AsnGlu 1760 1765 1770 His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu ValGlu Asp 1775 1780 1785 Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser ArgPro Tyr Ser 1790 1795 1800 Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu AspGln Arg Gln Gly 1805 1810 1815 Ala Glu Pro Arg Lys Asn Phe Val Lys ProAsn Glu Thr Lys Thr 1820 1825 1830 Tyr Phe Trp Lys Val Gln His His MetAla Pro Thr Lys Asp Glu 1835 1840 1845 Phe Asp Cys Lys Ala Trp Ala TyrPhe Ser Asp Val Asp Leu Glu 1850 1855 1860 Lys Asp Val His Ser Gly LeuIle Gly Pro Leu Leu Val Cys His 1865 1870 1875 Thr Asn Thr Leu Asn ProAla His Gly Arg Gln Val Thr Val Gln 1880 1885 1890 Glu Phe Ala Leu PhePhe Thr Ile Phe Asp Glu Thr Lys Ser Trp 1895 1900 1905 Tyr Phe Thr GluAsn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn 1910 1915 1920 Ile Gln MetGlu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His 1925 1930 1935 Ala IleAsn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met 1940 1945 1950 AlaGln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser 1955 1960 1965Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe Thr 1970 19751980 Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr 19851990 1995 Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly2000 2005 2010 Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His AlaGly 2015 2020 2025 Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys GlnThr Pro 2030 2035 2040 Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe GlnIle Thr Ala 2045 2050 2055 Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys LeuAla Arg Leu His 2060 2065 2070 Tyr Ser Gly Ser Ile Asn Ala Trp Ser ThrLys Glu Pro Phe Ser 2075 2080 2085 Trp Ile Lys Val Asp Leu Leu Ala ProMet Ile Ile His Gly Ile 2090 2095 2100 Lys Thr Gln Gly Ala Arg Gln LysPhe Ser Ser Leu Tyr Ile Ser 2105 2110 2115 Gln Phe Ile Ile Met Tyr SerLeu Asp Gly Lys Lys Trp Gln Thr 2120 2125 2130 Tyr Arg Gly Asn Ser ThrGly Thr Leu Met Val Phe Phe Gly Asn 2135 2140 2145 Val Asp Ser Ser GlyIle Lys His Asn Ile Phe Asn Pro Pro Ile 2150 2155 2160 Ile Ala Arg TyrIle Arg Leu His Pro Thr His Tyr Ser Ile Arg 2165 2170 2175 Ser Thr LeuArg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys 2180 2185 2190 Ser MetPro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln 2195 2200 2205 IleThr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser 2210 2215 2220Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp 2225 22302235 Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe 22402245 2250 Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys2255 2260 2265 Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile SerSer 2270 2275 2280 Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln AsnGly Lys 2285 2290 2295 Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe ThrPro Val Val 2300 2305 2310 Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg TyrLeu Arg Ile His 2315 2320 2325 Pro Gln Ser Trp Val His Gln Ile Ala LeuArg Met Glu Val Leu 2330 2335 2340 Gly Cys Glu Ala Gln Asp Leu Tyr 23452350 31 1471 DNA Homo sapiens 31 atggcgcccg tcgccgtctg ggccgcgctggccgtcggac tggagctctg ggctgcggcg 60 cacgccttgc ccgcccaggt ggcatttacaccctacgccc cggagcccgg gagcacatgc 120 cggctcagag aatactatga ccagacagctcagatgtgct gcagcaaatg ctcgccgggc 180 caacatgcaa aagtcttctg taccaagacctcggacaccg tgtgtgactc ctgtgaggac 240 agcacataca cccagctctg gaactgggttcccgagtgct tgagctgtgg ctcccgctgt 300 agctctgacc aggtggaaac tcaagcctgcactcgggaac agaaccgcat ctgcacctgc 360 aggcccggct ggtactgcgc gctgagcaagcaggaggggt gccggctgtg cgcgccgctg 420 cgcaagtgcc gcccgggctt cggcgtggccagaccaggaa ctgaaacatc agacgtggtg 480 tgcaagccct gtgccccggg gacgttctccaacacgactt catccacgga tatttgcagg 540 ccccaccaga tctgtaacgt ggtggccatccctgggaatg caagcatgga tgcagtctgc 600 acgtccacgt cccccacccg gagtatggccccaggggcag tacacttacc ccagccagtg 660 tccacacgat cccaacacac gcagccaactccagaaccca gcactgctcc aagcacctcc 720 ttcctgctcc caatgggccc cagccccccagctgaaggga gcactggcga cttcgctctt 780 ccagttggac tgattgtggg tgtgacagccttgggtctac taataatagg agtggtgaac 840 tgtgtcatca tgacccaggt gaaaaagaagcccttgtgcc tgcagagaga agccaaggtg 900 cctcacttgc ctgccgataa ggcccggggtacacagggcc ccgagcagca gcacctgctg 960 atcacagcgc cgagctccag cagcagctccctggagagct cggccagtgc gttggacaga 1020 agggcgccca ctcggaacca gccacaggcaccaggcgtgg aggccagtgg ggccggggag 1080 gcccgggcca gcaccgggag ctcagattcttcccctggtg gccatgggac ccaggtcaat 1140 gtcacctgca tcgtgaacgt ctgtagcagctctgaccaca gctcacagtg ctcctcccaa 1200 gccagctcca caatgggaga cacagattccagcccctcgg agtccccgaa ggacgagcag 1260 gtccccttct ccaaggagga atgtgcctttcggtcacagc tggagacgcc agagaccctg 1320 ctggggagca ccgaagagaa gcccctgccccttggagtgc ctgatgctgg gatgaagccc 1380 agttaaccag gccggtgtgg gctgtgtcgtagccaaggtg ggctgagccc tggcaggatg 1440 accctgcgaa ggggccctgg tccttccagg c1471 32 461 PRT Homo sapiens 32 Met Ala Pro Val Ala Val Trp Ala Ala LeuAla Val Gly Leu Glu Leu 1 5 10 15 Trp Ala Ala Ala His Ala Leu Pro AlaGln Val Ala Phe Thr Pro Tyr 20 25 30 Ala Pro Glu Pro Gly Ser Thr Cys ArgLeu Arg Glu Tyr Tyr Asp Gln 35 40 45 Thr Ala Gln Met Cys Cys Ser Lys CysSer Pro Gly Gln His Ala Lys 50 55 60 Val Phe Cys Thr Lys Thr Ser Asp ThrVal Cys Asp Ser Cys Glu Asp 65 70 75 80 Ser Thr Tyr Thr Gln Leu Trp AsnTrp Val Pro Glu Cys Leu Ser Cys 85 90 95 Gly Ser Arg Cys Ser Ser Asp GlnVal Glu Thr Gln Ala Cys Thr Arg 100 105 110 Glu Gln Asn Arg Ile Cys ThrCys Arg Pro Gly Trp Tyr Cys Ala Leu 115 120 125 Ser Lys Gln Glu Gly CysArg Leu Cys Ala Pro Leu Arg Lys Cys Arg 130 135 140 Pro Gly Phe Gly ValAla Arg Pro Gly Thr Glu Thr Ser Asp Val Val 145 150 155 160 Cys Lys ProCys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr 165 170 175 Asp IleCys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly 180 185 190 AsnAla Ser Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser 195 200 205Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser 210 215220 Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser 225230 235 240 Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser ThrGly 245 250 255 Asp Phe Ala Leu Pro Val Gly Leu Ile Val Gly Val Thr AlaLeu Gly 260 265 270 Leu Leu Ile Ile Gly Val Val Asn Cys Val Ile Met ThrGln Val Lys 275 280 285 Lys Lys Pro Leu Cys Leu Gln Arg Glu Ala Lys ValPro His Leu Pro 290 295 300 Ala Asp Lys Ala Arg Gly Thr Gln Gly Pro GluGln Gln His Leu Leu 305 310 315 320 Ile Thr Ala Pro Ser Ser Ser Ser SerSer Leu Glu Ser Ser Ala Ser 325 330 335 Ala Leu Asp Arg Arg Ala Pro ThrArg Asn Gln Pro Gln Ala Pro Gly 340 345 350 Val Glu Ala Ser Gly Ala GlyGlu Ala Arg Ala Ser Thr Gly Ser Ser 355 360 365 Asp Ser Ser Pro Gly GlyHis Gly Thr Gln Val Asn Val Thr Cys Ile 370 375 380 Val Asn Val Cys SerSer Ser Asp His Ser Ser Gln Cys Ser Ser Gln 385 390 395 400 Ala Ser SerThr Met Gly Asp Thr Asp Ser Ser Pro Ser Glu Ser Pro 405 410 415 Lys AspGlu Gln Val Pro Phe Ser Lys Glu Glu Cys Ala Phe Arg Ser 420 425 430 GlnLeu Glu Thr Pro Glu Thr Leu Leu Gly Ser Thr Glu Glu Lys Pro 435 440 445Leu Pro Leu Gly Val Pro Asp Ala Gly Met Lys Pro Ser 450 455 460 33 1475DNA Homo sapiens 33 tccacctgtc cccgcagcgc cggctcgcgc cctcctgccgcagccaccga gccgccgtct 60 agcgccccga cctcgccacc atgagagccc tgctggcgcgcctgcttctc tgcgtcctgg 120 tcgtgagcga ctccaaaggc agcaatgaac ttcatcaagttccatcgaac tgtgactgtc 180 taaatggagg aacatgtgtg tccaacaagt acttctccaacattcactgg tgcaactgcc 240 caaagaaatt cggagggcag cactgtgaaa tagataagtcaaaaacctgc tatgagggga 300 atggtcactt ttaccgagga aaggccagca ctgacaccatgggccggccc tgcctgccct 360 ggaactctgc cactgtcctt cagcaaacgt accatgcccacagatctgat gctcttcagc 420 tgggcctggg gaaacataat tactgcagga acccagacaaccggaggcga ccctggtgct 480 atgtgcaggt gggcctaaag ccgcttgtcc aagagtgcatggtgcatgac tgcgcagatg 540 gaaaaaagcc ctcctctcct ccagaagaat taaaatttcagtgtggccaa aagactctga 600 ggccccgctt taagattatt gggggagaat tcaccaccatcgagaaccag ccctggtttg 660 cggccatcta caggaggcac cgggggggct ctgtcacctacgtgtgtgga ggcagcctca 720 tcagcccttg ctgggtgatc agcgccacac actgcttcattgattaccca aagaaggagg 780 actacatcgt ctacctgggt cgctcaaggc ttaactccaacacgcaaggg gagatgaagt 840 ttgaggtgga aaacctcatc ctacacaagg actacagcgctgacacgctt gctcaccaca 900 acgacattgc cttgctgaag atccgttcca aggagggcaggtgtgcgcag ccatcccgga 960 ctatacagac catctgcctg ccctcgatgt ataacgatccccagtttggc acaagctgtg 1020 agatcactgg ctttggaaaa gagaattcta ccgactatctctatccggag cagctgaaga 1080 tgactgttgt gaagctgatt tcccaccggg agtgtcagcagccccactac tacggctctg 1140 aagtcaccac caaaatgctg tgtgctgctg acccacagtggaaaacagat tcctgccagg 1200 gagactcagg gggacccctc gtctgttccc tccaaggccgcatgactttg actggaattg 1260 tgagctgggg ccgtggatgt gccctgaagg acaagccaggcgtctacacg agagtctcac 1320 acttcttacc ctggatccgc agtcacacca aggaagagaatggcctggcc ctctgagggt 1380 ccccagggag gaaacgggca ccacccgctt tcttgctggttgtcattttt gcagtagagt 1440 catctccatc agctgtaaga agagactggg aagat 147534 431 PRT Homo sapiens 34 Met Arg Ala Leu Leu Ala Arg Leu Leu Leu CysVal Leu Val Val Ser 1 5 10 15 Asp Ser Lys Gly Ser Asn Glu Leu His GlnVal Pro Ser Asn Cys Asp 20 25 30 Cys Leu Asn Gly Gly Thr Cys Val Ser AsnLys Tyr Phe Ser Asn Ile 35 40 45 His Trp Cys Asn Cys Pro Lys Lys Phe GlyGly Gln His Cys Glu Ile 50 55 60 Asp Lys Ser Lys Thr Cys Tyr Glu Gly AsnGly His Phe Tyr Arg Gly 65 70 75 80 Lys Ala Ser Thr Asp Thr Met Gly ArgPro Cys Leu Pro Trp Asn Ser 85 90 95 Ala Thr Val Leu Gln Gln Thr Tyr HisAla His Arg Ser Asp Ala Leu 100 105 110 Gln Leu Gly Leu Gly Lys His AsnTyr Cys Arg Asn Pro Asp Asn Arg 115 120 125 Arg Arg Pro Trp Cys Tyr ValGln Val Gly Leu Lys Pro Leu Val Gln 130 135 140 Glu Cys Met Val His AspCys Ala Asp Gly Lys Lys Pro Ser Ser Pro 145 150 155 160 Pro Glu Glu LeuLys Phe Gln Cys Gly Gln Lys Thr Leu Arg Pro Arg 165 170 175 Phe Lys IleIle Gly Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp 180 185 190 Phe AlaAla Ile Tyr Arg Arg His Arg Gly Gly Ser Val Thr Tyr Val 195 200 205 CysGly Gly Ser Leu Ile Ser Pro Cys Trp Val Ile Ser Ala Thr His 210 215 220Cys Phe Ile Asp Tyr Pro Lys Lys Glu Asp Tyr Ile Val Tyr Leu Gly 225 230235 240 Arg Ser Arg Leu Asn Ser Asn Thr Gln Gly Glu Met Lys Phe Glu Val245 250 255 Glu Asn Leu Ile Leu His Lys Asp Tyr Ser Ala Asp Thr Leu AlaHis 260 265 270 His Asn Asp Ile Ala Leu Leu Lys Ile Arg Ser Lys Glu GlyArg Cys 275 280 285 Ala Gln Pro Ser Arg Thr Ile Gln Thr Ile Cys Leu ProSer Met Tyr 290 295 300 Asn Asp Pro Gln Phe Gly Thr Ser Cys Glu Ile ThrGly Phe Gly Lys 305 310 315 320 Glu Asn Ser Thr Asp Tyr Leu Tyr Pro GluGln Leu Lys Met Thr Val 325 330 335 Val Lys Leu Ile Ser His Arg Glu CysGln Gln Pro His Tyr Tyr Gly 340 345 350 Ser Glu Val Thr Thr Lys Met LeuCys Ala Ala Asp Pro Gln Trp Lys 355 360 365 Thr Asp Ser Cys Gln Gly AspSer Gly Gly Pro Leu Val Cys Ser Leu 370 375 380 Gln Gly Arg Met Thr LeuThr Gly Ile Val Ser Trp Gly Arg Gly Cys 385 390 395 400 Ala Leu Lys AspLys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu 405 410 415 Pro Trp IleArg Ser His Thr Lys Glu Glu Asn Gly Leu Ala Leu 420 425 430 35 107 PRTMus musculus 35 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala SerVal Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp ValAsn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro LysLeu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser ArgPhe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser SerLeu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His TyrThr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100105 36 120 PRT Mus musculus 36 Glu Val Gln Leu Val Glu Ser Gly Gly GlyLeu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala SerGly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala ProGly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly TyrThr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala AspThr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg AlaGlu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly PheTyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val SerSer 115 120 37 120 PRT Mus musculus 37 Gln Val Thr Leu Arg Glu Ser GlyPro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys ThrPhe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Ser Val Gly Trp IleArg Gln Pro Ser Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala Asp Ile Trp TrpAsp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr IleSer Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Lys Val Thr AsnMet Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Met IleThr Asn Trp Tyr Phe Asp Val Trp Gly Ala 100 105 110 Gly Thr Thr Val ThrVal Ser Ser 115 120 38 106 PRT Mus musculus 38 Asp Ile Gln Met Thr GlnSer Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr IleThr Cys Lys Cys Gln Leu Ser Val Gly Tyr Met 20 25 30 His Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr 35 40 45 Asp Thr Ser Lys LeuAla Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr GluPhe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala ThrTyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95 Phe Gly Gly GlyThr Lys Leu Glu Ile Lys 100 105 39 1039 DNA Homo sapiens 39 tcctgcacaggcagtgcctt gaagtgcttc ttcagagacc tttcttcata gactactttt 60 ttttctttaagcagcaaaag gagaaaattg tcatcaaagg atattccaga ttcttgacag 120 cattctcgtcatctctgagg acatcaccat catctcagga tgaggggcat gaagctgctg 180 ggggcgctgctggcactggc ggccctactg cagggggccg tgtccctgaa gatcgcagcc 240 ttcaacatccagacatttgg ggagaccaag atgtccaatg ccaccctcgt cagctacatt 300 gtgcagatcctgagccgcta tgacatcgcc ctggtccagg aggtcagaga cagccacctg 360 actgccgtggggaagctgct ggacaacctc aatcaggatg caccagacac ctatcactac 420 gtggtcagtgagccactggg acggaacagc tataaggagc gctacctgtt cgtgtacagg 480 cctgaccaggtgtctgcggt ggacagctac tactacgatg atggctgcga gccctgcggg 540 aacgacaccttcaaccgaga gccagccatt gtcaggttct tctcccggtt cacagaggtc 600 agggagtttgccattgttcc cctgcatgcg gccccggggg acgcagtagc cgagatcgac 660 gctctctatgacgtctacct ggatgtccaa gagaaatggg gcttggagga cgtcatgttg 720 atgggcgacttcaatgcggg ctgcagctat gtgagaccct cccagtggtc atccatccgc 780 ctgtggacaagccccacctt ccagtggctg atccccgaca gcgctgacac cacagctaca 840 cccacgcactgtgcctatga caggatcgtg gttgcaggga tgctgctccg aggcgccgtt 900 gttcccgactcggctcttcc ctttaacttc caggctgcct atggcctgag tgaccaactg 960 gcccaagccatcagtgacca ctatccagtg gaggtgatgc tgaagtgagc agcccctccc 1020 cacaccagttgaactgcag 1039 40 282 PRT Homo sapiens 40 Met Arg Gly Met Lys Leu LeuGly Ala Leu Leu Ala Leu Ala Ala Leu 1 5 10 15 Leu Gln Gly Ala Val SerLeu Lys Ile Ala Ala Phe Asn Ile Gln Thr 20 25 30 Phe Gly Glu Thr Lys MetSer Asn Ala Thr Leu Val Ser Tyr Ile Val 35 40 45 Gln Ile Leu Ser Arg TyrAsp Ile Ala Leu Val Gln Glu Val Arg Asp 50 55 60 Ser His Leu Thr Ala ValGly Lys Leu Leu Asp Asn Leu Asn Gln Asp 65 70 75 80 Ala Pro Asp Thr TyrHis Tyr Val Val Ser Glu Pro Leu Gly Arg Asn 85 90 95 Ser Tyr Lys Glu ArgTyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser 100 105 110 Ala Val Asp SerTyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn 115 120 125 Asp Thr PheAsn Arg Glu Pro Ala Ile Val Arg Phe Phe Ser Arg Phe 130 135 140 Thr GluVal Arg Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly 145 150 155 160Asp Ala Val Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val 165 170175 Gln Glu Lys Trp Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn 180185 190 Ala Gly Cys Ser Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu195 200 205 Trp Thr Ser Pro Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala AspThr 210 215 220 Thr Ala Thr Pro Thr His Cys Ala Tyr Asp Arg Ile Val ValAla Gly 225 230 235 240 Met Leu Leu Arg Gly Ala Val Val Pro Asp Ser AlaLeu Pro Phe Asn 245 250 255 Phe Gln Ala Ala Tyr Gly Leu Ser Asp Gln LeuAla Gln Ala Ile Ser 260 265 270 Asp His Tyr Pro Val Glu Val Met Leu Lys275 280 41 678 DNA Mus musculus 41 gacatcttgc tgactcagtc tccagccatcctgtctgtga gtccaggaga aagagtcagt 60 ttctcctgca gggccagtca gttcgttggctcaagcatcc actggtatca gcaaagaaca 120 aatggttctc caaggcttct cataaagtatgcttctgagt ctatgtctgg gatcccttcc 180 aggtttagtg gcagtggatc agggacagattttactctta gcatcaacac tgtggagtct 240 gaagatattg cagattatta ctgtcaacaaagtcatagct ggccattcac gttcggctcg 300 gggacaaatt tggaagtaaa agaagtgaagcttgaggagt ctggaggagg cttggtgcaa 360 cctggaggat ccatgaaact ctcctgtgttgcctctggat tcattttcag taaccactgg 420 atgaactggg tccgccagtc tccagagaaggggcttgagt gggttgctga aattagatca 480 aaatctatta attctgcaac acattatgcggagtctgtga aagggaggtt caccatctca 540 agagatgatt ccaaaagtgc tgtctacctgcaaatgaccg acttaagaac tgaagacact 600 ggcgtttatt actgttccag gaattactacggtagtacct acgactactg gggccaaggc 660 accactctca cagtctcc 678 42 226 PRTMus musculus 42 Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val SerPro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Phe ValGly Ser Ser 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro ArgLeu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Met Ser Gly Ile Pro Ser ArgPhe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn ThrVal Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser HisSer Trp Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Asn Leu Glu Val Lys GluVal Lys Leu Glu 100 105 110 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySer Met Lys Leu Ser 115 120 125 Cys Val Ala Ser Gly Phe Ile Phe Ser AsnHis Trp Met Asn Trp Val 130 135 140 Arg Gln Ser Pro Glu Lys Gly Leu GluTrp Val Ala Glu Ile Arg Ser 145 150 155 160 Lys Ser Ile Asn Ser Ala ThrHis Tyr Ala Glu Ser Val Lys Gly Arg 165 170 175 Phe Thr Ile Ser Arg AspAsp Ser Lys Ser Ala Val Tyr Leu Gln Met 180 185 190 Thr Asp Leu Arg ThrGlu Asp Thr Gly Val Tyr Tyr Cys Ser Arg Asn 195 200 205 Tyr Tyr Gly SerThr Tyr Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 210 215 220 Val Ser 22543 450 DNA Homo sapiens 43 gctgcatcag aagaggccat caagcacatc actgtccttctgccatggcc ctgtggatgc 60 gcctcctgcc cctgctggcg ctgctggccc tctggggacctgacccagcc gcagcctttg 120 tgaaccaaca cctgtgcggc tcacacctgg tggaagctctctacctagtg tgcggggaac 180 gaggcttctt ctacacaccc aagacccgcc gggaggcagaggacctgcag gtggggcagg 240 tggagctggg cgggggccct ggtgcaggca gcctgcagcccttggccctg gaggggtccc 300 tgcagaagcg tggcattgtg gaacaatgct gtaccagcatctgctccctc taccagctgg 360 agaactactg caactagacg cagcccgcag gcagccccccacccgccgcc tcctgcaccg 420 agagagatgg aataaagccc ttgaaccagc 450 44 110PRT Homo sapiens 44 Met Ala Leu Trp Met Arg Leu Leu Pro Leu Leu Ala LeuLeu Ala Leu 1 5 10 15 Trp Gly Pro Asp Pro Ala Ala Ala Phe Val Asn GlnHis Leu Cys Gly 20 25 30 Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys GlyGlu Arg Gly Phe 35 40 45 Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu AspLeu Gln Val Gly 50 55 60 Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly SerLeu Gln Pro Leu 65 70 75 80 Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly IleVal Glu Gln Cys Cys 85 90 95 Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu AsnTyr Cys Asn 100 105 110 45 1203 DNA Hepatitis B virus 45 atgggaggttggtcttccaa acctcgacaa ggcatgggga cgaatctttc tgttcccaat 60 cctctgggattctttcccga tcaccagttg gaccctgcgt tcggagccaa ctcaaacaat 120 ccagattgggacttcaaccc caacaaggat cactggccag aggcaatcaa ggtaggagcg 180 ggagacttcgggccagggtt caccccacca cacggcggtc ttttggggtg gagccctcag 240 gctcagggcatattgacaac agtgccagca gcgcctcctc ctgtttccac caatcggcag 300 tcaggaagacagcctactcc catctctcca cctctaagag acagtcatcc tcaggccatg 360 cagtggaactccacaacatt ccaccaagct ctgctagatc ccagagtgag gggcctatat 420 tttcctgctggtggctccag ttccggaaca gtaaaccctg ttccgactac tgtctcaccc 480 atatcgtcaatcttctcgag gactggggac cctgcaccga acatggagag cacaacatca 540 ggattcctaggacccctgct cgtgttacag gcggggtttt tcttgttgac aagaatcctc 600 acaataccacagagtctaga ctcgtggtgg acttctctca attttctagg gggagcaccc 660 acgtgtcctggccaaaattc gcagtcccca acctccaatc actcaccaac ctcttgtcct 720 ccaatttgtcctggttatcg ctggatgtgt ctgcggcgtt ttatcatatt cctcttcatc 780 ctgctgctatgcctcatctt cttgttggtt cttctggact accaaggtat gttgcccgtt 840 tgtcctctacttccaggaac atcaactacc agcacgggac catgcaagac ctgcacgatt 900 cctgctcaaggaacctctat gtttccctct tgttgctgta caaaaccttc ggacggaaac 960 tgcacttgtattcccatccc atcatcctgg gctttcgcaa gattcctatg ggagtgggcc 1020 tcagtccgtttctcctggct cagtttacta gtgccatttg ttcagtggtt cgcagggctt 1080 tcccccactgtttggctttc agttatatgg atgatgtggt attgggggcc aagtctgtac 1140 aacatcttgagtcccttttt acctctatta ccaattttct tttgtctttg ggtatacatt 1200 tga 1203 46400 PRT Hepatitis B virus 46 Met Gly Gly Trp Ser Ser Lys Pro Arg Gln GlyMet Gly Thr Asn Leu 1 5 10 15 Ser Val Pro Asn Pro Leu Gly Phe Phe ProAsp His Gln Leu Asp Pro 20 25 30 Ala Phe Gly Ala Asn Ser Asn Asn Pro AspTrp Asp Phe Asn Pro Asn 35 40 45 Lys Asp His Trp Pro Glu Ala Ile Lys ValGly Ala Gly Asp Phe Gly 50 55 60 Pro Gly Phe Thr Pro Pro His Gly Gly LeuLeu Gly Trp Ser Pro Gln 65 70 75 80 Ala Gln Gly Ile Leu Thr Thr Val ProAla Ala Pro Pro Pro Val Ser 85 90 95 Thr Asn Arg Gln Ser Gly Arg Gln ProThr Pro Ile Ser Pro Pro Leu 100 105 110 Arg Asp Ser His Pro Gln Ala MetGln Trp Asn Ser Thr Thr Phe His 115 120 125 Gln Ala Leu Leu Asp Pro ArgVal Arg Gly Leu Tyr Phe Pro Ala Gly 130 135 140 Gly Ser Ser Ser Gly ThrVal Asn Pro Val Pro Thr Thr Val Ser Pro 145 150 155 160 Ile Ser Ser IlePhe Ser Arg Thr Gly Asp Pro Ala Pro Asn Met Glu 165 170 175 Ser Thr ThrSer Gly Phe Leu Gly Pro Leu Leu Val Leu Gln Ala Gly 180 185 190 Phe PheLeu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu Asp Ser 195 200 205 TrpTrp Thr Ser Leu Asn Phe Leu Gly Gly Ala Pro Thr Cys Pro Gly 210 215 220Gln Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser Cys Pro 225 230235 240 Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe Ile Ile245 250 255 Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val LeuLeu 260 265 270 Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Leu Pro GlyThr Ser 275 280 285 Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Ile ProAla Gln Gly 290 295 300 Thr Ser Met Phe Pro Ser Cys Cys Cys Thr Lys ProSer Asp Gly Asn 305 310 315 320 Cys Thr Cys Ile Pro Ile Pro Ser Ser TrpAla Phe Ala Arg Phe Leu 325 330 335 Trp Glu Trp Ala Ser Val Arg Phe SerTrp Leu Ser Leu Leu Val Pro 340 345 350 Phe Val Gln Trp Phe Ala Gly LeuSer Pro Thr Val Trp Leu Ser Val 355 360 365 Ile Trp Met Met Trp Tyr TrpGly Pro Ser Leu Tyr Asn Ile Leu Ser 370 375 380 Pro Phe Leu Pro Leu LeuPro Ile Phe Phe Cys Leu Trp Val Tyr Ile 385 390 395 400 47 799 DNA Homosapiens 47 cgaaccactc agggtcctgt ggacagctca cctagctgca atggctacaggctcccggac 60 gtccctgctc ctggcttttg gcctgctctg cctgccctgg cttcaagagggcagtgcctt 120 cccaaccatt cccttatcca ggccttttga caacgctatg ctccgcgcccatcgtctgca 180 ccagctggcc tttgacacct accaggagtt tgaagaagcc tatatcccaaaggaacagaa 240 gtattcattc ctgcagaacc cccagacctc cctctgtttc tcagagtctattccgacacc 300 ctccaacagg gaggaaacac aacagaaatc caacctagag ctgctccgcatctccctgct 360 gctcatccag tcgtggctgg agcccgtgca gttcctcagg agtgtcttcgccaacagcct 420 ggtgtacggc gcctctgaca gcaacgtcta tgacctccta aaggacctagaggaaggcat 480 ccaaacgctg atggggaggc tggaagatgg cagcccccgg actgggcagatcttcaagca 540 gacctacagc aagttcgaca caaactcaca caacgatgac gcactactcaagaactacgg 600 gctgctctac tgcttcagga aggacatgga caaggtcgag acattcctgcgcatcgtgca 660 gtgccgctct gtggagggca gctgtggctt ctagctgccc gggtggcatccctgtgaccc 720 ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccaccagccttgtcct 780 aataaaatta agttgcatc 799 48 217 PRT Homo sapiens 48 MetAla Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu 1 5 10 15Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu 20 25 30Ser Arg Pro Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln 35 40 45Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys 50 55 60Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe 65 70 7580 Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys 85 9095 Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp 100105 110 Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val115 120 125 Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp LeuGlu 130 135 140 Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly SerPro Arg 145 150 155 160 Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys PheAsp Thr Asn Ser 165 170 175 His Asn Asp Asp Ala Leu Leu Lys Asn Tyr GlyLeu Leu Tyr Cys Phe 180 185 190 Arg Lys Asp Met Asp Lys Val Glu Thr PheLeu Arg Ile Val Gln Cys 195 200 205 Arg Ser Val Glu Gly Ser Cys Gly Phe210 215 49 963 DNA Homo sapiens 49 atggagacag acacactcct gttatgggtgctgctgctct gggttccagg ttccactggt 60 gacgtcaggc gagggccccg gagcctgcggggcagggacg cgccagcccc cacgccctgc 120 gtcccggccg agtgcttcga cctgctggtccgccactgcg tggcctgcgg gctcctgcgc 180 acgccgcggc cgaaaccggc cggggccagcagccctgcgc ccaggacggc gctgcagccg 240 caggagtcgg tgggcgcggg ggccggcgaggcggcggtcg acaaaactca cacatgccca 300 ccgtgcccag cacctgaact cctggggggaccgtcagtct tcctcttccc cccaaaaccc 360 aaggacaccc tcatgatctc ccggacccctgaggtcacat gcgtggtggt ggacgtgagc 420 cacgaagacc ctgaggtcaa gttcaactggtacgtggacg gcgtggaggt gcataatgcc 480 aagacaaagc cgcgggagga gcagtacaacagcacgtacc gtgtggtcag cgtcctcacc 540 gtcctgcacc aggactggct gaatggcaaggagtacaagt gcaaggtctc caacaaagcc 600 ctcccagccc ccatcgagaa aaccatctccaaagccaaag ggcagccccg agaaccacag 660 gtgtacaccc tgcccccatc ccgggatgagctgaccaaga accaggtcag cctgacctgc 720 ctggtcaaag gcttctatcc cagcgacatcgccgtggagt gggagagcaa tgggcagccg 780 gagaacaact acaagaccac gcctcccgtgttggactccg acggctcctt cttcctctac 840 agcaagctca ccgtggacaa gagcaggtggcagcagggga acgtcttctc atgctccgtg 900 atgcatgagg ctctgcacaa ccactacacgcagaagagcc tctccctgtc tcccgggaaa 960 tga 963 50 320 PRT Homo sapiens 50Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 1015 Gly Ser Thr Gly Asp Val Arg Arg Gly Pro Arg Ser Leu Arg Gly Arg 20 2530 Asp Ala Pro Ala Pro Thr Pro Cys Val Pro Ala Glu Cys Phe Asp Leu 35 4045 Leu Val Arg His Cys Val Ala Cys Gly Leu Leu Arg Thr Pro Arg Pro 50 5560 Lys Pro Ala Gly Ala Ser Ser Pro Ala Pro Arg Thr Ala Leu Gln Pro 65 7075 80 Gln Glu Ser Val Gly Ala Gly Ala Gly Glu Ala Ala Val Asp Lys Thr 8590 95 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser100 105 110 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile SerArg 115 120 125 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His GluAsp Pro 130 135 140 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu ValHis Asn Ala 145 150 155 160 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn SerThr Tyr Arg Val Val 165 170 175 Ser Val Leu Thr Val Leu His Gln Asp TrpLeu Asn Gly Lys Glu Tyr 180 185 190 Lys Cys Lys Val Ser Asn Lys Ala LeuPro Ala Pro Ile Glu Lys Thr 195 200 205 Ile Ser Lys Ala Lys Gly Gln ProArg Glu Pro Gln Val Tyr Thr Leu 210 215 220 Pro Pro Ser Arg Asp Glu LeuThr Lys Asn Gln Val Ser Leu Thr Cys 225 230 235 240 Leu Val Lys Gly PheTyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 245 250 255 Asn Gly Gln ProGlu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 260 265 270 Ser Asp GlySer Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 275 280 285 Arg TrpGln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 290 295 300 LeuHis Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 305 310 315320 51 107 PRT Homo sapiens 51 Asp Ile Gln Met Thr Gln Thr Pro Ser ThrLeu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg AlaSer Gln Asp Ile Asn Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro GlyLys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Thr Leu His Ser GlyVal Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr LeuThr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Phe CysGln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys ValGlu Val Lys 100 105 52 107 PRT Mus musculus 52 Asp Ile Gln Met Thr GlnThr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr IleSer Cys Arg Ala Ser Gln Asp Ile Asn Asn Tyr 20 25 30 Leu Asn Trp Tyr GlnGln Lys Pro Asp Gly Ile Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser ThrLeu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly ThrAsp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile AlaThr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly GlyGly Thr Lys Leu Glu Ile Lys 100 105 53 119 PRT Homo sapiens 53 Gln ValGln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 SerVal Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30 LeuIle Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 GlyVal Ile Tyr Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60 LysGly Arg Val Thr Leu Thr Val Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95Ala Arg Arg Asp Gly Asn Tyr Gly Trp Phe Ala Tyr Trp Gly Gln Gly 100 105110 Thr Leu Val Thr Val Ser Ser 115 54 119 PRT Mus musculus 54 Gln ValGln Leu Gln Gln Ser Gly Ala Glu Leu Val Gly Pro Gly Thr 1 5 10 15 SerVal Arg Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30 LeuIle Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 GlyVal Ile Tyr Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60 LysGly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys 85 90 95Ala Arg Arg Asp Gly Asn Tyr Gly Trp Phe Ala Tyr Trp Gly Arg Gly 100 105110 Thr Leu Val Thr Val Ser Ala 115 55 214 PRT Homo sapiens 55 Asp IleGln Met Thr Gln Thr Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 AspArg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Asn Asn Tyr 20 25 30 LeuAsn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 TyrTyr Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 SerGly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80Asp Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Val Lys Arg Thr Val Ala Ala 100 105110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn SerGln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr TyrSer Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu LysHis Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser SerPro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 56 448 PRTHomo sapiens 56 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys ProGly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala PheThr Asn Tyr 20 25 30 Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly LeuGlu Trp Ile 35 40 45 Gly Val Ile Tyr Pro Gly Ser Gly Gly Thr Asn Tyr AsnGlu Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Val Asp Glu Ser Thr AsnThr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr AlaVal Tyr Phe Cys 85 90 95 Ala Arg Arg Asp Gly Asn Tyr Gly Trp Phe Ala TyrTrp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr LysGly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr SerGly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe ProGlu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr SerGly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu TyrSer Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly ThrGln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr LysVal Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His ThrCys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 SerVal Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly LysGlu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala ProIle Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu ProGln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys AsnGln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser AspIle Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn TyrLys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe PheLeu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln GlyAsn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn HisTyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 57 8540 DNA Homosapiens 57 gacgtcgcgg ccgctctagg cctccaaaaa agcctcctca ctacttctggaatagctcag 60 aggccgaggc ggcctcggcc tctgcataaa taaaaaaaat tagtcagccatgcatggggc 120 ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttaggggcgggact 180 atggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctggggagcctggg 240 gactttccac acctggttgc tgactaattg agatgcatgc tttgcatacttctgcctgct 300 ggggagcctg gggactttcc acaccctaac tgacacacat tccacagaattaattcccct 360 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatatggagttccgc 420 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgacccccgcccattg 480 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttccattgacgtcaa 540 tgggtggact atttacggta aactgcccac ttggcagtac atcaagtgtatcatatgcca 600 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcattatgcccagtac 660 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcatcgctattacc 720 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttgactcacgggga 780 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcaccaaaatcaacgg 840 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcggtaggcgtgta 900 cggtgggagg tctatataag cagagctggg tacgtgaacc gtcagatcgcctggagacgc 960 catcacagat ctctcaccat gagggtcccc gctcagctcc tggggctcctgctgctctgg 1020 ctcccaggtg cacgatgtga tggtaccaag gtggaaatca aacgtacggtggctgcacca 1080 tctgtcttca tcttcccgcc atctgatgag cagttgaaat ctggaactgcctctgttgtg 1140 tgcctgctga ataacttcta tcccagagag gccaaagtac agtggaaggtggataacgcc 1200 ctccaatcgg gtaactccca ggagagtgtc acagagcagg acagcaaggacagcacctac 1260 agcctcagca gcaccctgac gctgagcaaa gcagactacg agaaacacaaagtctacgcc 1320 tgcgaagtca cccatcaggg cctgagctcg cccgtcacaa agagcttcaacaggggagag 1380 tgttgaattc agatccgtta acggttacca actacctaga ctggattcgtgacaacatgc 1440 ggccgtgata tctacgtatg atcagcctcg actgtgcctt ctagttgccagccatctgtt 1500 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccactgtcctttcc 1560 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctattctggggggt 1620 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggcatgctggggat 1680 gcggtgggct ctatggaacc agctggggct cgacagctat gccaagtacgccccctattg 1740 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgaccttatgggact 1800 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtgatgcggtttt 1860 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttccaagtctccacc 1920 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggactttccaaaatgtc 1980 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgggaggtctata 2040 taagcagagc tgggtacgtc ctcacattca gtgatcagca ctgaacacagacccgtcgac 2100 atgggttgga gcctcatctt gctcttcctt gtcgctgttg ctacgcgtgtcgctagcacc 2160 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgggggcacagcg 2220 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtcgtggaactca 2280 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctcaggactctac 2340 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagacctacatctgc 2400 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagcagagcccaaatcttgt 2460 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctggggggaccgtcagtc 2520 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccctgaggtcaca 2580 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactggtacgtggac 2640 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaacagcacgtac 2700 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaaggactacaag 2760 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctccaaagccaaa 2820 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatgagctgaccagg 2880 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacatcgccgtggag 2940 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgtgctggactcc 3000 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtggcagcagggg 3060 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacacgcagaagagc 3120 ctctccctgt ctccgggtaa atgaggatcc gttaacggtt accaactacctagactggat 3180 tcgtgacaac atgcggccgt gatatctacg tatgatcagc ctcgactgtgccttctagtt 3240 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaaggtgccactc 3300 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagtaggtgtcatt 3360 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaagacaatagca 3420 ggcatgctgg ggatgcggtg ggctctatgg aaccagctgg ggctcgacagcgctggatct 3480 cccgatcccc agctttgctt ctcaatttct tatttgcata atgagaaaaaaaggaaaatt 3540 aattttaaca ccaattcagt agttgattga gcaaatgcgt tgccaaaaaggatgctttag 3600 agacagtgtt ctctgcacag ataaggacaa acattattca gagggagtacccagagctga 3660 gactcctaag ccagtgagtg gcacagcatt ctagggagaa atatgcttgtcatcaccgaa 3720 gcctgattcc gtagagccac accttggtaa gggccaatct gctcacacaggatagagagg 3780 gcaggagcca gggcagagca tataaggtga ggtaggatca gttgctcctcacatttgctt 3840 ctgacatagt tgtgttggga gcttggatag cttggacagc tcagggctgcgatttcgcgc 3900 caaacttgac ggcaatccta gcgtgaaggc tggtaggatt ttatccccgctgccatcatg 3960 gttcgaccat tgaactgcat cgtcgccgtg tcccaaaata tggggattggcaagaacgga 4020 gacctaccct ggcctccgct caggaacgag ttcaagtact tccaaagaatgaccacaacc 4080 tcttcagtgg aaggtaaaca gaatctggtg attatgggta ggaaaacctggttctccatt 4140 cctgagaaca atcgaccttt aaaggacaga attaatatag ttctcagtagagaactcaaa 4200 gaaccaccac gaggagctca ttttcttgcc aaaagtttgg atgatgccttaagacttatt 4260 gaacaaccgg aattggcaag taaagtagac atggtttgga tagtcggaggcagttctgtt 4320 taccaggaag ccatgaatca accaggccac cttagactct ttgtgacaaggatcatgcag 4380 gaatttgaaa gtgacacgtt tttcccagaa attgatttgg ggaaatataaacttctccca 4440 gaatacccag gcgtcctctc tgaggtccag gaggaaaaag gcatcaagtataagtttgaa 4500 gtctacgaga agaaagacta acaggaagat gctttcaagt tctctgctcccctcctaaag 4560 tcatgcattt ttataagacc atgggacttt tgctggcttt agatcagcctcgactgtgcc 4620 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttgaccctggaagg 4680 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcattgtctgagtag 4740 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggaggattgggaaga 4800 caatagcagg catgctgggg atgcggtggg ctctatggaa ccagctggggctcgagctac 4860 tagctttgct tctcaatttc ttatttgcat aatgagaaaa aaaggaaaattaattttaac 4920 accaattcag tagttgattg agcaaatgcg ttgccaaaaa ggatgctttagagacagtgt 4980 tctctgcaca gataaggaca aacattattc agagggagta cccagagctgagactcctaa 5040 gccagtgagt ggcacagcat tctagggaga aatatgcttg tcatcaccgaagcctgattc 5100 cgtagagcca caccttggta agggccaatc tgctcacaca ggatagagagggcaggagcc 5160 agggcagagc atataaggtg aggtaggatc agttgctcct cacatttgcttctgacatag 5220 ttgtgttggg agcttggatc gatcctctat ggttgaacaa gatggattgcacgcaggttc 5280 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacagacaatcggctg 5340 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttctttttgtcaagac 5400 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctatcgtggctggc 5460 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgggaagggactg 5520 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttgctcctgccga 5580 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatccggctacctg 5640 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcggatggaagccgg 5700 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccagccgaactgtt 5760 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgacccatggcgatgc 5820 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcgactgtggccg 5880 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgatattgctgaaga 5940 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccgcttcccgatt 6000 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcgggactctggggtt 6060 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgattccaccgccgc 6120 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctggatgatcctcca 6180 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattgcagcttataa 6240 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttttttcactgca 6300 ttctagttgt ggtttgtcca aactcatcaa tctatcttat catgtctggatcgcggccgc 6360 gatcccgtcg agagcttggc gtaatcatgg tcatagctgt ttcctgtgtgaaattgttat 6420 ccgctcacaa ttccacacaa catacgagcc ggagcataaa gtgtaaagcctggggtgcct 6480 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttccagtcgggaa 6540 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggcggtttgcgta 6600 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgttcggctgcggc 6660 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatcaggggataacg 6720 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaaaaggccgcgt 6780 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaatcgacgctcaa 6840 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccccctggaagct 6900 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtccgcctttctcc 6960 cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagttcggtgtagg 7020 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgaccgctgcgcct 7080 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcgccactggcag 7140 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctacagagttcttga 7200 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgcgctctgctga 7260 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaaaccaccgctg 7320 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaaggatctcaag 7380 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaactcacgttaag 7440 ggattttggt catgagatta tcaaaaagga tcttcaccta gatccttttaaattaaaaat 7500 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagttaccaatgct 7560 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccatagttgcctgac 7620 tccccgtcgt gtagataact acgatacggg agggcttacc atctggccccagtgctgcaa 7680 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaaccagccagccg 7740 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccagtctattaatt 7800 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaacgttgttgcca 7860 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattcagctccggtt 7920 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcggttagctcct 7980 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactcatggttatgg 8040 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttctgtgactggtg 8100 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgctcttgcccgg 8160 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctcatcattggaa 8220 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatccagttcgatgt 8280 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagcgtttctgggt 8340 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgacacggaaatgtt 8400 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggttattgtctca 8460 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggttccgcgcacat 8520 ttccccgaaa agtgccacct 8540 58 9209 DNA Mus musculus 58gacgtcgcgg ccgctctagg cctccaaaaa agcctcctca ctacttctgg aatagctcag 60aggccgaggc ggcctcggcc tctgcataaa taaaaaaaat tagtcagcca tgcatggggc 120ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggact 180atggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg 240gactttccac acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct 300ggggagcctg gggactttcc acaccctaac tgacacacat tccacagaat taattcccct 360agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc 420gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg 480acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa 540tgggtggact atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca 600agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac 660atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc 720atggtgatgc ggttttggca gtacatcaat gggcgtggat accggtttga ctcacgcgga 780tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg 840gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta 900cggtgggagg tctatataag cagagctggg tacgtgaacc gtcagatcgc ctggagacgc 960catcacagat ctctcactat ggattttcag gtgcagatta tcagcttcct gctaatcagt 1020gcttcagtca taatgtccag aggacaaatt gttctctccc agtctccagc aatcctgtct 1080gcatctccag gggagaaggt cacaatgact tgcagggcca gctcaagtgt aagttacatc 1140cactggttcc agcagaagcc aggatcctcc cccaaaccct ggatttatgc cacatccaac 1200ctggcttctg gagtccctgt tcgcttcagt ggcagtgggt ctgggacttc ttactctctc 1260acaatcagca gagtggaggc tgaagatgct gccacttatt actgccagca gtggactagt 1320aacccaccca cgttcggagg ggggaccaag ctggaaatca aacgtacggt ggctgcacca 1380tctgtcttca tcttcccgcc atctgatgag cagttgaaat ctggaactgc ctctgttgtg 1440tgcctgctga ataacttcta tcccagagag gccaaagtac agtggaaggt ggataacgcc 1500ctccaatcgg gtaactccca ggagagtgtc acagagcagg acagcaagga cagcacctac 1560agcctcagca gcaccctgac gctgagcaaa gcagactacg agaaacacaa agtctacgcc 1620tgcgaagtca cccatcaggg cctgagctcg cccgtcacaa agagcttcaa caggggagag 1680tgttgaattc agatccgtta acggttacca actacctaga ctggattcgt gacaacatgc 1740ggccgtgata tctacgtatg atcagcctcg actgtgcctt ctagttgcca gccatctgtt 1800gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc 1860taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt 1920ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat 1980gcggtgggct ctatggaacc agctggggct cgacagctat gccaagtacg ccccctattg 2040acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact 2100ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt 2160ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc 2220ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc 2280gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata 2340taagcagagc tgggtacgtc ctcacattca gtgatcagca ctgaacacag acccgtcgac 2400atgggttgga gcctcatctt gctcttcctt gtcgctgttg ctacgcgtgt cctgtcccag 2460gtacaactgc agcagcctgg ggctgagctg gtgaagcctg gggcctcagt gaagatgtcc 2520tgcaaggctt ctggctacac atttaccagt tacaatatgc actgggtaaa acagacacct 2580ggtcggggcc tggaatggat tggagctatt tatcccggaa atggtgatac ttcctacaat 2640cagaagttca aaggcaaggc cacattgact gcagacaaat cctccagcac agcctacatg 2700cagctcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag atcgacttac 2760tacggcggtg actggtactt caatgtctgg ggcgcaggga ccacggtcac cgtctctgca 2820gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 2880ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 2940tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 3000ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 3060tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agcagagccc 3120aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 3180ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 3240gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 3300tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 3360agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 3420gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 3480aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 3540ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 3600gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 3660ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 3720cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 3780cagaagagcc tctccctgtc tccgggtaaa tgaggatccg ttaacggtta ccaactacct 3840agactggatt cgtgacaaca tgcggccgtg atatctacgt atgatcagcc tcgactgtgc 3900cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 3960gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 4020ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 4080acaatagcag gcatgctggg gatgcggtgg gctctatgga accagctggg gctcgacagc 4140gctggatctc ccgatcccca gctttgcttc tcaatttctt atttgcataa tgagaaaaaa 4200aggaaaatta attttaacac caattcagta gttgattgag caaatgcgtt gccaaaaagg 4260atgctttaga gacagtgttc tctgcacaga taaggacaaa cattattcag agggagtacc 4320cagagctgag actcctaagc cagtgagtgg cacagcattc tagggagaaa tatgcttgtc 4380atcaccgaag cctgattccg tagagccaca ccttggtaag ggccaatctg ctcacacagg 4440atagagaggg caggagccag ggcagagcat ataaggtgag gtaggatcag ttgctcctca 4500catttgcttc tgacatagtt gtgttgggag cttggatagc ttggacagct cagggctgcg 4560atttcgcgcc aaacttgacg gcaatcctag cgtgaaggct ggtaggattt tatccccgct 4620gccatcatgg ttcgaccatt gaactgcatc gtcgccgtgt cccaaaatat ggggattggc 4680aagaacggag acctaccctg gcctccgctc aggaacgagt tcaagtactt ccaaagaatg 4740accacaacct cttcagtgga aggtaaacag aatctggtga ttatgggtag gaaaacctgg 4800ttctccattc ctgagaagaa tcgaccttta aaggacagaa ttaatatagt tctcagtaga 4860gaactcaaag aaccaccacg aggagctcat tttcttgcca aaagtttgga tgatgcctta 4920agacttattg aacaaccgga attggcaagt aaagtagaca tggtttggat agtcggaggc 4980agttctgttt accaggaagc catgaatcaa ccaggccacc ttagactctt tgtgacaagg 5040atcatgcagg aatttgaaag tgacacgttt ttcccagaaa ttgatttggg gaaatataaa 5100cttctcccag aatacccagg cgtcctctct gaggtccagg aggaaaaagg catcaagtat 5160aagtttgaag tctacgagaa gaaagactaa caggaagatg ctttcaagtt ctctgctccc 5220ctcctaaagc tatgcatttt tataagacca tgggactttt gctggcttta gatcagcctc 5280gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac 5340cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg 5400tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga 5460ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggaac cagctggggc 5520tcgagctact agctttgctt ctcaatttct tatttgcata atgagaaaaa aaggaaaatt 5580aattttaaca ccaattcagt agttgattga gcaaatgcgt tgccaaaaag gatgctttag 5640agacagtgtt ctctgcacag ataaggacaa acattattca gagggagtac ccagagctga 5700gactcctaag ccagtgagtg gcacagcatt ctagggagaa atatgcttgt catcaccgaa 5760gcctgattcc gtagagccac accttggtaa gggccaatct gctcacacag gatagagagg 5820gcaggagcca gggcagagca tataaggtga ggtaggatca gttgctcctc acatttgctt 5880ctgacatagt tgtgttggga gcttggatcg atcctctatg gttgaacaag atggattgca 5940cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac 6000aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt 6060tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc 6120gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg 6180aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc 6240tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc 6300ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat 6360ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc 6420cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca 6480tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga 6540ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat 6600tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc 6660tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact 6720ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc 6780accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg 6840atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca 6900gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 6960tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatc tatcttatca tgtctggatc 7020gcggccgcga tcccgtcgag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa 7080attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 7140ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc 7200agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 7260gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 7320ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 7380gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 7440aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 7500gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 7560ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 7620cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt 7680cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 7740gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 7800cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 7860agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg 7920ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 7980ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 8040gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 8100cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 8160attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 8220accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 8280ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 8340gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 8400agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 8460ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 8520ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 8580gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 8640ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 8700tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 8760tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 8820cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 8880tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 8940gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 9000tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 9060ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 9120attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 9180cgcgcacatt tccccgaaaa gtgccacct 9209 59 384 DNA Mus musculus 59atggattttc aggtgcagat tatcagcttc ctgctaatca gtgcttcagt cataatgtcc 60agagggcaaa ttgttctctc ccagtctcca gcaatcctgt ctgcatctcc aggggagaag 120gtcacaatga cttgcagggc cagctcaagt gtaagttaca tccactggtt ccagcagaag 180ccaggatcct cccccaaacc ctggatttat gccacatcca acctggcttc tggagtccct 240gttcgcttca gtggcagtgg gtctgggact tcttactctc tcacaatcag cagagtggag 300gctgaagatg ctgccactta ttactgccag cagtggacta gtaacccacc cacgttcgga 360ggggggacca agctggaaat caaa 384 60 128 PRT Mus musculus 60 Met Asp PheGln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val IleMet Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu SerAla Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser SerVal Ser Tyr Ile His Trp Phe Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro LysPro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 ValArg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 SerArg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110Thr Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 115 120125 61 420 DNA Mus musculus 61 atgggttgga gcctcatctt gctcttccttgtcgctgttg ctacgcgtgt cctgtcccag 60 gtacaactgc agcagcctgg ggctgagctggtgaagcctg gggcctcagt gaagatgtcc 120 tgcaaggctt ctggctacac atttaccagttacaatatgc actgggtaaa acagacacct 180 ggtcggggcc tggaatggat tggagctatttatcccggaa atggtgatac ttcctacaat 240 cagaagttca aaggcaaggc cacattgactgcagacaaat cctccagcac agcctacatg 300 cagctcagca gcctgacatc tgaggactctgcggtctatt actgtgcaag atcgacttac 360 tacggcggtg actggtactt caatgtctggggcgcaggga ccacggtcac cgtctctgca 420 62 140 PRT Mus musculus 62 Met GlyTrp Ser Leu Ile Leu Leu Phe Leu Val Ala Val Ala Thr Arg 1 5 10 15 ValLeu Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys 20 25 30 ProGly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 ThrSer Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu 50 55 60 GluTrp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn 65 70 75 80Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 85 90 95Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105110 Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn 115120 125 Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala 130 135 140 631395 DNA Homo sapiens 63 atgtattcca atgtgatagg aactgtaacc tctggaaaaaggaaggttta tcttttgtcc 60 ttgctgctca ttggcttctg ggactgcgtg acctgtcacgggagccctgt ggacatctgc 120 acagccaagc cgcgggacat tcccatgaat cccatgtgcatttaccgctc cccggagaag 180 aaggcaactg aggatgaggg ctcagaacag aagatcccggaggccaccaa ccggcgtgtc 240 tgggaactgt ccaaggccaa ttcccgcttt gctaccactttctatcagca cctggcagat 300 tccaagaatg acaatgataa cattttcctg tcacccctgagtatctccac ggcttttgct 360 atgaccaagc tgggtgcctg taatgacacc ctccagcaactgatggaggt atttaagttt 420 gacaccatat ctgagaaaac atctgatcag atccacttcttctttgccaa actgaactgc 480 cgactctatc gaaaagccaa caaatcctcc aagttagtatcagccaatcg cctttttgga 540 gacaaatccc ttaccttcaa tgagacctac caggacatcagtgagttggt atatggagcc 600 aagctccagc ccctggactt caaggaaaat gcagagcaatccagagcggc catcaacaaa 660 tgggtgtcca ataagaccga aggccgaatc accgatgtcattccctcgga agccatcaat 720 gagctcactg ttctggtgct ggttaacacc atttacttcaagggcctgtg gaagtcaaag 780 ttcagccctg agaacacaag gaaggaactg ttctacaaggctgatggaga gtcgtgttca 840 gcatctatga tgtaccagga aggcaagttc cgttatcggcgcgtggctga aggcacccag 900 gtgcttgagt tgcccttcaa aggtgatgac atcaccatggtcctcatctt gcccaagcct 960 gagaagagcc tggccaaggt ggagaaggaa ctcaccccagaggtgctgca ggagtggctg 1020 gatgaattgg aggagatgat gctggtggtc cacatgccccgcttccgcat tgaggacggc 1080 ttcagtttga aggagcagct gcaagacatg ggccttgtcgatctgttcag ccctgaaaag 1140 tccaaactcc caggtattgt tgcagaaggc cgagatgacctctatgtctc agatgcattc 1200 cataaggcat ttcttgaggt aaatgaagaa ggcagtgaagcagctgcaag taccgctgtt 1260 gtgattgctg gccgttcgct aaaccccaac agggtgactttcaaggccaa caggcctttc 1320 ctggttttta taagagaagt tcctctgaac actattatcttcatgggcag agtagccaac 1380 ccttgtgtta agtaa 1395 64 464 PRT Homo sapiens64 Met Tyr Ser Asn Val Ile Gly Thr Val Thr Ser Gly Lys Arg Lys Val 1 510 15 Tyr Leu Leu Ser Leu Leu Leu Ile Gly Phe Trp Asp Cys Val Thr Cys 2025 30 His Gly Ser Pro Val Asp Ile Cys Thr Ala Lys Pro Arg Asp Ile Pro 3540 45 Met Asn Pro Met Cys Ile Tyr Arg Ser Pro Glu Lys Lys Ala Thr Glu 5055 60 Asp Glu Gly Ser Glu Gln Lys Ile Pro Glu Ala Thr Asn Arg Arg Val 6570 75 80 Trp Glu Leu Ser Lys Ala Asn Ser Arg Phe Ala Thr Thr Phe Tyr Gln85 90 95 His Leu Ala Asp Ser Lys Asn Asp Asn Asp Asn Ile Phe Leu Ser Pro100 105 110 Leu Ser Ile Ser Thr Ala Phe Ala Met Thr Lys Leu Gly Ala CysAsn 115 120 125 Asp Thr Leu Gln Gln Leu Met Glu Val Phe Lys Phe Asp ThrIle Ser 130 135 140 Glu Lys Thr Ser Asp Gln Ile His Phe Phe Phe Ala LysLeu Asn Cys 145 150 155 160 Arg Leu Tyr Arg Lys Ala Asn Lys Ser Ser LysLeu Val Ser Ala Asn 165 170 175 Arg Leu Phe Gly Asp Lys Ser Leu Thr PheAsn Glu Thr Tyr Gln Asp 180 185 190 Ile Ser Glu Leu Val Tyr Gly Ala LysLeu Gln Pro Leu Asp Phe Lys 195 200 205 Glu Asn Ala Glu Gln Ser Arg AlaAla Ile Asn Lys Trp Val Ser Asn 210 215 220 Lys Thr Glu Gly Arg Ile ThrAsp Val Ile Pro Ser Glu Ala Ile Asn 225 230 235 240 Glu Leu Thr Val LeuVal Leu Val Asn Thr Ile Tyr Phe Lys Gly Leu 245 250 255 Trp Lys Ser LysPhe Ser Pro Glu Asn Thr Arg Lys Glu Leu Phe Tyr 260 265 270 Lys Ala AspGly Glu Ser Cys Ser Ala Ser Met Met Tyr Gln Glu Gly 275 280 285 Lys PheArg Tyr Arg Arg Val Ala Glu Gly Thr Gln Val Leu Glu Leu 290 295 300 ProPhe Lys Gly Asp Asp Ile Thr Met Val Leu Ile Leu Pro Lys Pro 305 310 315320 Glu Lys Ser Leu Ala Lys Val Glu Lys Glu Leu Thr Pro Glu Val Leu 325330 335 Gln Glu Trp Leu Asp Glu Leu Glu Glu Met Met Leu Val Val His Met340 345 350 Pro Arg Phe Arg Ile Glu Asp Gly Phe Ser Leu Lys Glu Gln LeuGln 355 360 365 Asp Met Gly Leu Val Asp Leu Phe Ser Pro Glu Lys Ser LysLeu Pro 370 375 380 Gly Ile Val Ala Glu Gly Arg Asp Asp Leu Tyr Val SerAsp Ala Phe 385 390 395 400 His Lys Ala Phe Leu Glu Val Asn Glu Glu GlySer Glu Ala Ala Ala 405 410 415 Ser Thr Ala Val Val Ile Ala Gly Arg SerLeu Asn Pro Asn Arg Val 420 425 430 Thr Phe Lys Ala Asn Arg Pro Phe LeuVal Phe Ile Arg Glu Val Pro 435 440 445 Leu Asn Thr Ile Ile Phe Met GlyArg Val Ala Asn Pro Cys Val Lys 450 455 460 65 1962 DNA Homo sapiens 65atgcgtcccc tgcgcccccg cgccgcgctg ctggcgctcc tggcctcgct cctggccgcg 60cccccggtgg ccccggccga ggccccgcac ctggtgcagg tggacgcggc ccgcgcgctg 120tggcccctgc ggcgcttctg gaggagcaca ggcttctgcc ccccgctgcc acacagccag 180gctgaccagt acgtcctcag ctgggaccag cagctcaacc tcgcctatgt gggcgccgtc 240cctcaccgcg gcatcaagca ggtccggacc cactggctgc tggagcttgt caccaccagg 300gggtccactg gacggggcct gagctacaac ttcacccacc tggacgggta cttggacctt 360ctcagggaga accagctcct cccagggttt gagctgatgg gcagcgcctc gggccacttc 420actgactttg aggacaagca gcaggtgttt gagtggaagg acttggtctc cagcctggcc 480aggagataca tcggtaggta cggactggcg catgtttcca agtggaactt cgagacgtgg 540aatgagccag accaccacga ctttgacaac gtctccatga ccatgcaagg cttcctgaac 600tactacgatg cctgctcgga gggtctgcgc gccgccagcc ccgccctgcg gctgggaggc 660cccggcgact ccttccacac cccaccgcga tccccgctga gctggggcct cctgcgccac 720tgccacgacg gtaccaactt cttcactggg gaggcgggcg tgcggctgga ctacatctcc 780ctccacagga agggtgcgcg cagctccatc tccatcctgg agcaggagaa ggtcgtcgcg 840cagcagatcc ggcagctctt ccccaagttc gcggacaccc ccatttacaa cgacgaggcg 900gacccgctgg tgggctggtc cctgccacag ccgtggaggg cggacgtgac ctacgcggcc 960atggtggtga aggtcatcgc gcagcatcag aacctgctac tggccaacac cacctccgcc 1020ttcccctacg cgctcctgag caacgacaat gccttcctga gctaccaccc gcaccccttc 1080gcgcagcgca cgctcaccgc gcgcttccag gtcaacaaca cccgcccgcc gcacgtgcag 1140ctgttgcgca agccggtgct cacggccatg gggctgctgg cgctgctgga tgaggagcag 1200ctctgggccg aagtgtcgca ggccgggacc gtcctggaca gcaaccacac ggtgggcgtc 1260ctggccagcg cccaccgccc ccagggcccg gccgacgcct ggcgcgccgc ggtgctgatc 1320tacgcgagcg acgacacccg cgcccacccc aaccgcagcg tcgcggtgac cctgcggctg 1380cgcggggtgc cccccggccc gggcctggtc tacgtcacgc gctacctgga caacgggctc 1440tgcagccccg acggcgagtg gcggcgcctg ggccggcccg tcttccccac ggcagagcag 1500ttccggcgca tgcgcgcggc tgaggacccg gtggccgcgg cgccccgccc cttacccgcc 1560ggcggccgcc tgaccctgcg ccccgcgctg cggctgccgt cgcttttgct ggtgcacgtg 1620tgtgcgcgcc ccgagaagcc gcccgggcag gtcacgcggc tccgcgccct gcccctgacc 1680caagggcagc tggttctggt ctggtcggat gaacacgtgg gctccaagtg cctgtggaca 1740tacgagatcc agttctctca ggacggtaag gcgtacaccc cggtcagcag gaagccatcg 1800accttcaacc tctttgtgtt cagcccagac acaggtgctg tctctggctc ctaccgagtt 1860cgagccctgg actactgggc ccgaccaggc cccttctcgg accctgtgcc gtacctggag 1920gtccctgtgc caagagggcc cccatccccg ggcaatccat ga 1962 66 653 PRT Homosapiens 66 Met Arg Pro Leu Arg Pro Arg Ala Ala Leu Leu Ala Leu Leu AlaSer 1 5 10 15 Leu Leu Ala Ala Pro Pro Val Ala Pro Ala Glu Ala Pro HisLeu Val 20 25 30 Gln Val Asp Ala Ala Arg Ala Leu Trp Pro Leu Arg Arg PheTrp Arg 35 40 45 Ser Thr Gly Phe Cys Pro Pro Leu Pro His Ser Gln Ala AspGln Tyr 50 55 60 Val Leu Ser Trp Asp Gln Gln Leu Asn Leu Ala Tyr Val GlyAla Val 65 70 75 80 Pro His Arg Gly Ile Lys Gln Val Arg Thr His Trp LeuLeu Glu Leu 85 90 95 Val Thr Thr Arg Gly Ser Thr Gly Arg Gly Leu Ser TyrAsn Phe Thr 100 105 110 His Leu Asp Gly Tyr Leu Asp Leu Leu Arg Glu AsnGln Leu Leu Pro 115 120 125 Gly Phe Glu Leu Met Gly Ser Ala Ser Gly HisPhe Thr Asp Phe Glu 130 135 140 Asp Lys Gln Gln Val Phe Glu Trp Lys AspLeu Val Ser Ser Leu Ala 145 150 155 160 Arg Arg Tyr Ile Gly Arg Tyr GlyLeu Ala His Val Ser Lys Trp Asn 165 170 175 Phe Glu Thr Trp Asn Glu ProAsp His His Asp Phe Asp Asn Val Ser 180 185 190 Met Thr Met Gln Gly PheLeu Asn Tyr Tyr Asp Ala Cys Ser Glu Gly 195 200 205 Leu Arg Ala Ala SerPro Ala Leu Arg Leu Gly Gly Pro Gly Asp Ser 210 215 220 Phe His Thr ProPro Arg Ser Pro Leu Ser Trp Gly Leu Leu Arg His 225 230 235 240 Cys HisAsp Gly Thr Asn Phe Phe Thr Gly Glu Ala Gly Val Arg Leu 245 250 255 AspTyr Ile Ser Leu His Arg Lys Gly Ala Arg Ser Ser Ile Ser Ile 260 265 270Leu Glu Gln Glu Lys Val Val Ala Gln Gln Ile Arg Gln Leu Phe Pro 275 280285 Lys Phe Ala Asp Thr Pro Ile Tyr Asn Asp Glu Ala Asp Pro Leu Val 290295 300 Gly Trp Ser Leu Pro Gln Pro Trp Arg Ala Asp Val Thr Tyr Ala Ala305 310 315 320 Met Val Val Lys Val Ile Ala Gln His Gln Asn Leu Leu LeuAla Asn 325 330 335 Thr Thr Ser Ala Phe Pro Tyr Ala Leu Leu Ser Asn AspAsn Ala Phe 340 345 350 Leu Ser Tyr His Pro His Pro Phe Ala Gln Arg ThrLeu Thr Ala Arg 355 360 365 Phe Gln Val Asn Asn Thr Arg Pro Pro His ValGln Leu Leu Arg Lys 370 375 380 Pro Val Leu Thr Ala Met Gly Leu Leu AlaLeu Leu Asp Glu Glu Gln 385 390 395 400 Leu Trp Ala Glu Val Ser Gln AlaGly Thr Val Leu Asp Ser Asn His 405 410 415 Thr Val Gly Val Leu Ala SerAla His Arg Pro Gln Gly Pro Ala Asp 420 425 430 Ala Trp Arg Ala Ala ValLeu Ile Tyr Ala Ser Asp Asp Thr Arg Ala 435 440 445 His Pro Asn Arg SerVal Ala Val Thr Leu Arg Leu Arg Gly Val Pro 450 455 460 Pro Gly Pro GlyLeu Val Tyr Val Thr Arg Tyr Leu Asp Asn Gly Leu 465 470 475 480 Cys SerPro Asp Gly Glu Trp Arg Arg Leu Gly Arg Pro Val Phe Pro 485 490 495 ThrAla Glu Gln Phe Arg Arg Met Arg Ala Ala Glu Asp Pro Val Ala 500 505 510Ala Ala Pro Arg Pro Leu Pro Ala Gly Gly Arg Leu Thr Leu Arg Pro 515 520525 Ala Leu Arg Leu Pro Ser Leu Leu Leu Val His Val Cys Ala Arg Pro 530535 540 Glu Lys Pro Pro Gly Gln Val Thr Arg Leu Arg Ala Leu Pro Leu Thr545 550 555 560 Gln Gly Gln Leu Val Leu Val Trp Ser Asp Glu His Val GlySer Lys 565 570 575 Cys Leu Trp Thr Tyr Glu Ile Gln Phe Ser Gln Asp GlyLys Ala Tyr 580 585 590 Thr Pro Val Ser Arg Lys Pro Ser Thr Phe Asn LeuPhe Val Phe Ser 595 600 605 Pro Asp Thr Gly Ala Val Ser Gly Ser Tyr ArgVal Arg Ala Leu Asp 610 615 620 Tyr Trp Ala Arg Pro Gly Pro Phe Ser AspPro Val Pro Tyr Leu Glu 625 630 635 640 Val Pro Val Pro Arg Gly Pro ProSer Pro Gly Asn Pro 645 650 67 1290 DNA Homo sapiens 67 atgcagctgaggaacccaga actacatctg ggctgcgcgc ttgcgcttcg cttcctggcc 60 ctcgtttcctgggacatccc tggggctaga gcactggaca atggattggc aaggacgcct 120 accatgggctggctgcactg ggagcgcttc atgtgcaacc ttgactgcca ggaagagcca 180 gattcctgcatcagtgagaa gctcttcatg gagatggcag agctcatggt ctcagaaggc 240 tggaaggatgcaggttatga gtacctctgc attgatgact gttggatggc tccccaaaga 300 gattcagaaggcagacttca ggcagaccct cagcgctttc ctcatgggat tcgccagcta 360 gctaattatgttcacagcaa aggactgaag ctagggattt atgcagatgt tggaaataaa 420 acctgcgcaggcttccctgg gagttttgga tactacgaca ttgatgccca gacctttgct 480 gactggggagtagatctgct aaaatttgat ggttgttact gtgacagttt ggaaaatttg 540 gcagatggttataagcacat gtccttggcc ctgaatagga ctggcagaag cattgtgtac 600 tcctgtgagtggcctcttta tatgtggccc tttcaaaagc ccaattatac agaaatccga 660 cagtactgcaatcactggcg aaattttgct gacattgatg attcctggaa aagtataaag 720 agtatcttggactggacatc ttttaaccag gagagaattg ttgatgttgc tggaccaggg 780 ggttggaatgacccagatat gttagtgatt ggcaactttg gcctcagctg gaatcagcaa 840 gtaactcagatggccctctg ggctatcatg gctgctcctt tattcatgtc taatgacctc 900 cgacacatcagccctcaagc caaagctctc cttcaggata aggacgtaat tgccatcaat 960 caggaccccttgggcaagca agggtaccag cttagacagg gagacaactt tgaagtgtgg 1020 gaacgacctctctcaggctt agcctgggct gtagctatga taaaccggca ggagattggt 1080 ggacctcgctcttataccat cgcagttgct tccctgggta aaggagtggc ctgtaatcct 1140 gcctgcttcatcacacagct cctccctgtg aaaaggaagc tagggttcta tgaatggact 1200 tcaaggttaagaagtcacat aaatcccaca ggcactgttt tgcttcagct agaaaataca 1260 atgcagatgtcattaaaaga cttactttaa 1290 68 429 PRT Homo sapiens 68 Met Gln Leu ArgAsn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu 1 5 10 15 Arg Phe LeuAla Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu 20 25 30 Asp Asn GlyLeu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu 35 40 45 Arg Phe MetCys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile 50 55 60 Ser Glu LysLeu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly 65 70 75 80 Trp LysAsp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met 85 90 95 Ala ProGln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg 100 105 110 PhePro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly 115 120 125Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly 130 135140 Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala 145150 155 160 Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys AspSer 165 170 175 Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu AlaLeu Asn 180 185 190 Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp ProLeu Tyr Met 195 200 205 Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile ArgGln Tyr Cys Asn 210 215 220 His Trp Arg Asn Phe Ala Asp Ile Asp Asp SerTrp Lys Ser Ile Lys 225 230 235 240 Ser Ile Leu Asp Trp Thr Ser Phe AsnGln Glu Arg Ile Val Asp Val 245 250 255 Ala Gly Pro Gly Gly Trp Asn AspPro Asp Met Leu Val Ile Gly Asn 260 265 270 Phe Gly Leu Ser Trp Asn GlnGln Val Thr Gln Met Ala Leu Trp Ala 275 280 285 Ile Met Ala Ala Pro LeuPhe Met Ser Asn Asp Leu Arg His Ile Ser 290 295 300 Pro Gln Ala Lys AlaLeu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn 305 310 315 320 Gln Asp ProLeu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn 325 330 335 Phe GluVal Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala 340 345 350 MetIle Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile Ala 355 360 365Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile 370 375380 Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr 385390 395 400 Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu LeuGln 405 410 415 Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu 420425 69 351 DNA Homo sapiens 69 atggattact acagaaaata tgcagctatctttctggtca cattgtcggt gtttctgcat 60 gttctccatt ccgctcctga tgtgcaggattgcccagaat gcacgctaca ggaaaaccca 120 ttcttctccc agccgggtgc cccaatacttcagtgcatgg gctgctgctt ctctagagca 180 tatcccactc cactaaggtc caagaagacgatgttggtcc aaaagaacgt cacctcagag 240 tccacttgct gtgtagctaa atcatataacagggtcacag taatgggggg tttcaaagtg 300 gagaaccaca cggcgtgcca ctgcagtacttgttattatc acaaatctta a 351 70 116 PRT Homo sapiens 70 Met Asp Tyr TyrArg Lys Tyr Ala Ala Ile Phe Leu Val Thr Leu Ser 1 5 10 15 Val Phe LeuHis Val Leu His Ser Ala Pro Asp Val Gln Asp Cys Pro 20 25 30 Glu Cys ThrLeu Gln Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala Pro 35 40 45 Ile Leu GlnCys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro 50 55 60 Leu Arg SerLys Lys Thr Met Leu Val Gln Lys Asn Val Thr Ser Glu 65 70 75 80 Ser ThrCys Cys Val Ala Lys Ser Tyr Asn Arg Val Thr Val Met Gly 85 90 95 Gly PheLys Val Glu Asn His Thr Ala Cys His Cys Ser Thr Cys Tyr 100 105 110 TyrHis Lys Ser 115 71 498 DNA Homo sapiens 71 atggagatgt tccaggggctgctgctgttg ctgctgctga gcatgggcgg gacatgggca 60 tccaaggagc cgcttcggccacggtgccgc cccatcaatg ccaccctggc tgtggagaag 120 gagggctgcc ccgtgtgcatcaccgtcaac accaccatct gtgccggcta ctgccccacc 180 atgacccgcg tgctgcagggggtcctgccg gccctgcctc aggtggtgtg caactaccgc 240 gatgtgcgct tcgagtccatccggctccct ggctgcccgc gcggcgtgaa ccccgtggtc 300 tcctacgccg tggctctcagctgtcaatgt gcactctgcc gccgcagcac cactgactgc 360 gggggtccca aggaccaccccttgacctgt gatgaccccc gcttccagga ctcctcttcc 420 tcaaaggccc ctccccccagccttccaagc ccatcccgac tcccggggcc ctcggacacc 480 ccgatcctcc cacaataa 49872 165 PRT Homo sapiens 72 Met Glu Met Phe Gln Gly Leu Leu Leu Leu LeuLeu Leu Ser Met Gly 1 5 10 15 Gly Thr Trp Ala Ser Lys Glu Pro Leu ArgPro Arg Cys Arg Pro Ile 20 25 30 Asn Ala Thr Leu Ala Val Glu Lys Glu GlyCys Pro Val Cys Ile Thr 35 40 45 Val Asn Thr Thr Ile Cys Ala Gly Tyr CysPro Thr Met Thr Arg Val 50 55 60 Leu Gln Gly Val Leu Pro Ala Leu Pro GlnVal Val Cys Asn Tyr Arg 65 70 75 80 Asp Val Arg Phe Glu Ser Ile Arg LeuPro Gly Cys Pro Arg Gly Val 85 90 95 Asn Pro Val Val Ser Tyr Ala Val AlaLeu Ser Cys Gln Cys Ala Leu 100 105 110 Cys Arg Arg Ser Thr Thr Asp CysGly Gly Pro Lys Asp His Pro Leu 115 120 125 Thr Cys Asp Asp Pro Arg PheGln Asp Ser Ser Ser Ser Lys Ala Pro 130 135 140 Pro Pro Ser Leu Pro SerPro Ser Arg Leu Pro Gly Pro Ser Asp Thr 145 150 155 160 Pro Ile Leu ProGln 165 73 165 PRT Homo sapiens 73 Ala Pro Pro Arg Leu Ile Cys Asp SerArg Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu Ala Glu AsnIle Thr Thr Gly Cys Ala Glu His 20 25 30 Cys Ser Leu Asn Glu Asn Ile ThrVal Pro Asp Thr Lys Val Asn Phe 35 40 45 Tyr Ala Trp Lys Arg Met Glu ValGly Gln Gln Ala Val Glu Val Trp 50 55 60 Gln Gly Leu Ala Leu Leu Ser GluAla Val Leu Arg Gly Gln Ala Leu 65 70 75 80 Leu Val Asn Ser Ser Gln ProTrp Glu Pro Leu Gln Leu His Val Asp 85 90 95 Lys Ala Val Ser Gly Leu ArgSer Leu Thr Thr Leu Leu Arg Ala Leu 100 105 110 Gly Ala Gln Lys Glu AlaIle Ser Pro Pro Asp Ala Ala Ser Ala Ala 115 120 125 Pro Leu Arg Thr IleThr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val 130 135 140 Tyr Ser Asn PheLeu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys ArgThr Gly Asp 165 74 588 DNA Homo sapiens 74 atggccctcc tgttccctctactggcagcc ctagtgatga ccagctatag ccctgttgga 60 tctctgggct gtgatctgcctcagaaccat ggcctactta gcaggaacac cttggtgctt 120 ctgcaccaaa tgaggagaatctcccctttc ttgtgtctca aggacagaag agacttcagg 180 ttcccccagg agatggtaaaagggagccag ttgcagaagg cccatgtcat gtctgtcctc 240 catgagatgc tgcagcagatcttcagcctc ttccacacag agcgctcctc tgctgcctgg 300 aacatgaccc tcctagaccaactccacact ggacttcatc agcaactgca acacctggag 360 acctgcttgc tgcaggtagtgggagaagga gaatctgctg gggcaattag cagccctgca 420 ctgaccttga ggaggtacttccagggaatc cgtgtctacc tgaaagagaa gaaatacagc 480 gactgtgcct gggaagttgtcagaatggaa atcatgaaat ccttgttctt atcaacaaac 540 atgcaagaaa gactgagaagtaaagataga gacctgggct catcttga 588 75 195 PRT Homo sapiens 75 Met AlaLeu Leu Phe Pro Leu Leu Ala Ala Leu Val Met Thr Ser Tyr 1 5 10 15 SerPro Val Gly Ser Leu Gly Cys Asp Leu Pro Gln Asn His Gly Leu 20 25 30 LeuSer Arg Asn Thr Leu Val Leu Leu His Gln Met Arg Arg Ile Ser 35 40 45 ProPhe Leu Cys Leu Lys Asp Arg Arg Asp Phe Arg Phe Pro Gln Glu 50 55 60 MetVal Lys Gly Ser Gln Leu Gln Lys Ala His Val Met Ser Val Leu 65 70 75 80His Glu Met Leu Gln Gln Ile Phe Ser Leu Phe His Thr Glu Arg Ser 85 90 95Ser Ala Ala Trp Asn Met Thr Leu Leu Asp Gln Leu His Thr Gly Leu 100 105110 His Gln Gln Leu Gln His Leu Glu Thr Cys Leu Leu Gln Val Val Gly 115120 125 Glu Gly Glu Ser Ala Gly Ala Ile Ser Ser Pro Ala Leu Thr Leu Arg130 135 140 Arg Tyr Phe Gln Gly Ile Arg Val Tyr Leu Lys Glu Lys Lys TyrSer 145 150 155 160 Asp Cys Ala Trp Glu Val Val Arg Met Glu Ile Met LysSer Leu Phe 165 170 175 Leu Ser Thr Asn Met Gln Glu Arg Leu Arg Ser LysAsp Arg Asp Leu 180 185 190 Gly Ser Ser 195

What is claimed:
 1. A cell-free, in vitro method of remodeling agranulocyte colony stimulating factor (G-CSF) peptide, said peptidehaving the formula:

wherein AA is a terminal or internal amino acid residue of said peptide;X¹-X² is a saccharide covalently linked to said AA, wherein X¹ is afirst glycosyl residue; and X² is a second glycosyl residue covalentlylinked to X¹, wherein X¹ and X² are selected from monosaccharyl andoligosaccharyl residues; said method comprising: (a) removing X² or asaccharyl subunit thereof from said peptide, thereby forming a truncatedglycan; and (b) contacting said truncated glycan with at least oneglycosyltransferase and at least one glycosyl donor under conditionssuitable to transfer said at least one glycosyl donor to said truncatedglycan, thereby remodeling said G-CSF peptide.
 2. The method of claim 1,further comprising: (c) removing X¹, thereby exposing said AA; and (d)contacting said AA with at least one glycosyltransferase and at leastone glycosyl donor under conditions suitable to transfer said at leastone glycosyl donor to said AA, thereby remodeling said G-CSF peptide. 3.The method of claim 1, further comprising: (e) prior to step (b),removing a group added to said saccharide during post-translationalmodification.
 4. The method of claim 3, wherein said group is a memberselected from phosphate, sulfate, carboxylate and esters thereof.
 5. Themethod of claim 1, wherein said peptide has the formula:

wherein Z is a member selected from O, S, NH and a crosslinker.
 6. Themethod of claim 1, wherein at least one of said glycosyl donorscomprises a modifying group.
 7. The method of claim 1, wherein saidmodifying group is a member selected from the group consisting of apolymer, a therapeutic moiety, a detectable label, a reactive linkergroup, a targeting moiety, and a peptide.
 8. The method of claim 7,wherein said modifying group is a water soluble polymer.
 9. The methodof claim 8, wherein said water soluble polymer comprises poly(ethyleneglycol).
 10. The method of claim 9, wherein said poly(ethylene glycol)has a molecular weight distribution that is essentially homodisperse.11. A cell-free in vitro method of remodeling a G-CSF peptide, saidpeptide having the formula:

wherein X³, X⁴, X⁵, X⁶, X⁷, and X¹⁷ are independently selectedmonosaccharyl or oligosaccharyl residues; and a, b, c, d, e and x areindependently selected from the integers 0, 1 and 2, with the provisothat at least one member selected from a, b, c, d, and e and x are 1 or2; said method comprising: (a) removing at least one of X³, X⁴, X⁵, X⁶,X⁷, or X¹⁷, a saccharyl subunit thereof from said peptide, therebyforming a truncated glycan; and (b) contacting said truncated glycanwith at least one glycosyltransferase and at least one glycosyl donorunder conditions suitable to transfer said at least one glycosyl donorto said truncated glycan, thereby remodeling said G-CSF peptide.
 12. Themethod of claim 11, wherein said removing of step (a) produces atruncated glycan in which a, b, c, e and x are each
 0. 13. The method ofclaim 11, wherein X³, X⁵, and X⁷, are selected from the group consistingof (mannose)_(z) and (mannose)_(z)-(X⁸)_(y) wherein X⁸ is a glycosylmoiety selected from mono- and oligo-saccharides; y is an integerselected from 0 and 1; and z is an integer between 1 and 20, whereinwhen z is 3 or greater, (mannose)_(z) is selected from linear andbranched structures.
 14. The method of claim 11, wherein X⁴ is selectedfrom the group consisting of GlcNAc and xylose.
 15. The method of claim11, wherein X³, X⁵, and X⁷ are (mannose)_(u), wherein u is selected fromthe integers between 1 and 20, and when u is 3 or greater, (mannose)_(u)is selected from linear and branched structures.
 16. The method of claim11, wherein at least one of said glycosyl donors comprises a modifyinggroup.
 17. The method of claim 16, wherein said modifying group is amember selected from the group consisting of a polymer, a therapeuticmoiety, a detectable label, a reactive linker group, a targeting moiety,and a peptide.
 18. The method of claim 17 wherein said modifying groupis a water soluble polymer.
 19. The method of claim 18, wherein saidwater soluble polymer comprises poly(ethylene glycol).
 20. The method ofclaim 19, wherein said poly(ethylene glycol) has a molecular weightdistribution that is essentially homodisperse.
 21. A cell-free in vitromethod of remodeling a G-CSF peptide comprising a glycan having theformula:

wherein r, s, and t are integers independently selected from 0 and 1,said method comprising: (a) contacting said peptide with at least oneglycosyltransferase and at least one glycosyl donor under conditionssuitable to transfer said at least one glycosyl donor to said glycan,thereby remodeling said G-CSF peptide.
 22. The method of claim 21,wherein at least one of said glycosyl donors comprises a modifyinggroup.
 23. The method of claim 22, wherein said modifying group is amember selected from the group consisting of a polymer, a therapeuticmoiety, a detectable label, a reactive linker group, a targeting moiety,and a peptide.
 24. The method of claim 23 wherein said modifying groupis a water soluble polymer.
 25. The method of claim 24, wherein saidwater soluble polymer comprises poly(ethylene glycol).
 26. The method ofclaim 25, wherein said poly(ethylene glycol) has a molecular weightdistribution that is essentially homodisperse.
 27. The method of claim1, wherein said peptide has the formula:

wherein X⁹ and X¹⁰ are independently selected monosaccharyl oroligosaccharyl residues; and m, n and f are integers selected from 0and
 1. 28. The method of claim 1, wherein said peptide has the formula:

wherein X¹¹ and X¹² are independently selected glycosyl moieties; and rand x are integers independently selected from 0 and
 1. 29. The methodof claim 28, wherein X¹¹ and X¹² are (mannose)_(q), wherein q isselected from the integers between 1 and 20, and when q is three orgreater, (mannose)_(q) is selected from linear and branched structures.30. The method of claim 1, wherein said peptide has the formula:

wherein X¹³, X¹⁴, and X¹⁵ are independently selected glycosyl residues;and g, h, i, j, k, and p are independently selected from the integers 0and 1, with the proviso that at least one of g, h, i, j, k and p is 1.31. The method of claim 30, wherein X¹⁴ and X¹⁵ are membersindependently selected from GlcNAc and Sia; and i and k areindependently selected from the integers 0 and 1, with the proviso thatat least one of i and k is 1 and if k is 1, g, h and j are
 0. 32. Themethod of claim 1, wherein said peptide has the formula:

wherein X¹⁶ is a member selected from:

wherein s and i are integers independently selected from 0 and
 1. 33.The method of claim 1, wherein said removing utilizes a glycosidase. 34.A cell-free, in vitro method of remodeling a G-CSF peptide having theformula:

wherein AA is a terminal or internal amino acid residue of said peptide;X¹ is a glycosyl residue covalently linked to said AA, selected frommonosaccharyl and oligosaccharyl residues; and u is an integer selectedfrom 0 and 1, said method comprising: contacting said peptide with atleast one glycosyltransferase and at least one glycosyl donor underconditions suitable to transfer said at least one glycosyl donor to saidtruncated glycan, wherein said glycosyl donor comprises a modifyinggroup, thereby remodeling said G-CSF peptide.
 35. The method of claim34, wherein said modifying group is a member selected from the groupconsisting of a polymer, a therapeutic moiety, a detectable label, areactive linker group, a targeting moiety, and a peptide.
 36. The methodof claim 35 wherein said modifying group is a water soluble polymer. 37.The method of claim 36, wherein said water soluble polymer comprisespoly(ethylene glycol).
 38. The method of claim 37, wherein saidpoly(ethylene glycol) has a molecular weight distribution that isessentially homodisperse.
 39. A covalent conjugate between a G-CSFpeptide and a modifying group that alters a property of said peptide,wherein said modifying group is covalently attached to said peptide at apreselected glycosyl or amino acid residue of said peptide via an intactglycosyl linking group.
 40. The covalent conjugate of claim 39, whereinsaid modifying group is a member selected from the group consisting of apolymer, a therapeutic moiety, a detectable label, a reactive linkergroup, a targeting moiety, and a peptide.
 41. The covalent conjugate ofclaim 39, wherein said modifying group and an intact glycosyl linkinggroup precursor are linked as a covalently attached unit to said peptidevia the action of an enzyme, said enzyme converting said precursor tosaid intact glycosyl linking group, thereby forming said conjugate. 42.The covalent conjugate of claim 39 comprising: a first modifying groupcovalently linked to a first residue of said peptide via a first intactglycosyl linking group, and a second glycosyl linking group linked to asecond residue of said peptide via a second intact glycosyl linkinggroup.
 43. The covalent conjugate of claim 42, wherein said firstresidue and said second residue are structurally identical.
 44. Thecovalent conjugate of claim 42, wherein said first residue and saidsecond residue have different structures.
 45. The covalent conjugate ofclaim 42 wherein said first residue and said second residue are glycosylresidues.
 46. The covalent conjugate of claim 42, wherein said firstresidue and said second residue are amino acid residues.
 47. Thecovalent conjugate of claim 39, wherein said peptide is remodeled priorto forming said conjugate.
 48. The covalent conjugate of claim 47,wherein the remodeled peptide is remodeled to introduce an acceptormoiety for said intact glycosyl linking group.
 49. The covalentconjugate of claim 39, wherein said modifying group is a water-solublepolymer.
 50. The covalent conjugate of claim 49, wherein saidwater-soluble polymer comprises poly(ethylene glycol).
 51. The covalentconjugate of claim 39, wherein said intact glycosyl linking unit is amember selected from the group consisting of a sialic acid residue, aGal residue, a GlcNAc residue and a GalNAc residue.
 52. The covalentconjugate of claim 50, wherein said poly(ethylene glycol) has amolecular weight distribution that is essentially homodisperse.
 53. Amethod of forming a covalent conjugate between a polymer and aglycosylated or non-glycosylated peptide, wherein said polymer isconjugated to said peptide via an intact glycosyl linking groupinterposed between and covalently linked to both said peptide and saidpolymer, said method comprising: contacting said peptide with a mixturecomprising a nucleotide sugar covalently linked to said polymer and aglycosyltransferase for which said nucleotide sugar is a substrate underconditions sufficient to form said conjugate, wherein said peptide isG-CSF.
 54. The method of claim 53, wherein said polymer is awater-soluble polymer.
 55. The method of claim 53, wherein said glycosyllinking group is covalently attached to a glycosyl residue covalentlyattached to said peptide.
 56. The method of claim 53, wherein saidglycosyl linking group is covalently attached to an amino acid residueof said peptide.
 57. The method of claim 53, wherein said polymercomprises a member selected from the group consisting of a polyalkyleneoxide and a polypeptide.
 58. The method of claim 57, wherein saidpolyalkylene oxide is poly(ethylene glycol).
 59. The method of claim 58,wherein said poly(ethylene glycol) has a degree of polymerization offrom about 1 to about 20,000.
 60. The method of claim 59, wherein saidpolyethylene glycol has a degree of polymerization of from about 1 toabout 5,000.
 61. The method of claim 60, wherein said polyethyleneglycol has a degree of polymerization of from about 1 to about 1,000.62. The method of claim 53, wherein said glycosyltransferase is selectedfrom the group consisting of sialyltransferase, galactosyltransferase,glucosyltransferase, GalNAc transferase, GlcNAc transferase,fucosyltransferase, and mannosyltransferase.
 63. The method of claim 53,wherein said glycosyltransferase is recombinantly produced.
 64. Themethod of claim 63, wherein said glycosyltransferase is a recombinantprokaryotic enzyme.
 65. The method of claim 63, wherein saidglycosyltransferase is a recombinant eukaryotic enzyme.
 66. The methodof claim 53, wherein said nucleotide sugar is selected from the groupconsisting of UDP-glycoside, CMP-glycoside, and GDP-glycoside.
 67. Themethod of claim 66, wherein said nucleotide sugar is selected from thegroup consisting of UDP-galactose, UDP-galactosamine, UDP-glucose,UDP-glucosamine, UDP-N-acetylgalactosamine, UDP-N-acetylglucosamine,GDP-mannose, GDP-fucose, CMP-sialic acid, CMP-NeuAc.
 68. The method ofclaim 53, wherein said glycosylated peptide is partially deglycosylatedprior to said contacting.
 69. The method of claim 53, wherein saidintact glycosyl linking group is a sialic acid residue.
 70. The methodof claim 53, wherein said method is performed in a cell-freeenvironment.
 71. The method of claim 53, wherein said covalent conjugateis isolated.
 72. The method of claim 71, wherein said covalent conjugateis isolated by membrane filtration.
 73. A composition for forming aconjugate between a peptide and a modified sugar, said compositioncomprising: an admixture of a modified sugar, a glycosyltransferase, anda peptide acceptor substrate, wherein said modified sugar has covalentlyattached thereto a member selected from a polymer, a therapeutic moietyand a biomolecule, wherein said peptide is G-CSF.
 74. A G-CSF peptideremodeled by the method of claim
 1. 75. A pharmaceutical compositioncomprising the G-CSF peptide of claim
 74. 76. A G-CSF peptide remodeledby the method of claim
 11. 77. A pharmaceutical composition comprisingthe G-CSF peptide of claim
 76. 78. A G-CSF peptide remodeled by themethod of claim
 21. 79. A pharmaceutical composition comprising theG-CSF peptide of claim
 78. 80. A G-CSF peptide remodeled by the methodof claim
 27. 81. A pharmaceutical composition comprising the G-CSFpeptide of claim
 80. 82. A G-CSF peptide remodeled by the method ofclaim
 28. 83. A pharmaceutical composition comprising the G-CSF peptideof claim
 82. 84. A G-CSF peptide remodeled by the method of claim 34.85. A pharmaceutical composition comprising the G-CSF peptide of claim84.
 86. A cell-free, in vitro method of remodeling a peptide having theformula:

wherein AA is a terminal or internal amino acid residue of said peptide,said method comprising: contacting said peptide with at least oneglycosyltransferase and at least one glycosyl donor under conditionssuitable to transfer said at least one glycosyl donor to said amino acidresidue, wherein said glycosyl donor comprises a modifying group,thereby remodeling said peptide, wherein said peptide is G-CSF.
 87. Amethod of forming a conjugate between a G-CSF peptide and a modifyinggroup, wherein said modifying group is covalently attached to said G-CSFpeptide through an intact glycosyl linking group, said G-CSF peptidecomprising a glycosyl residue having the formula:

wherein a, b, c, and e are members independently selected from 0 and 1;d is 0; and R is a modifying group, a sialic acid or an oligosialicacid, said method comprising: (a) contacting said G-CSF peptide with aglycosyltransferase and a modified glycosyl donor, comprising a glycosylmoiety which is a substrate for said glycosyltransferase covalentlylinked to said modifying group, under conditions appropriate for theformation of said intact glycosyl linking group.
 88. The method of claim87, further comprising: (b) prior to step (a), contacting said G-CSFpeptide with a sialidase under conditions appropriate to remove sialicacid from said G-CSF peptide.
 89. The method of claim 87, furthercomprising: (c) prior to step (a), contacting said G-CSF peptide with agalactosyl transferase and a galactose donor under conditionsappropriate to transfer said galactose to said G-CSF peptide.
 90. Themethod of claim 87, further comprising: (d) contacting the product fromstep (a) with a moiety that reacts with said modifying group, therebyforming a conjugate between said intact glycosyl linking group and saidmoiety.
 91. The method of claim 87, further comprising: (e) prior tostep (a), contacting said G-CSF peptide with N-acetylgalactosaminetransferase and a GalNAc donor under conditions appropriate to transferGalNAc to said G-CSF peptide.
 92. The method of claim 87, furthercomprising: (f) prior to step (a), contacting said G-CSF peptide withendo-N-acetylgalactosaminidase operating synthetically and a GalNAcdonor under conditions appropriate to transfer GalNAc to said G-CSFpeptide.
 93. The method of claim 87, wherein said modifying group is amember selected from a polymer, a toxin, a radioisotope, a therapeuticmoiety and a glycoconjugate.
 94. The method of claim 87, wherein a, b,c, and e are
 0. 95. The method of claim 87, wherein a and e are membersindependently selected from 0 and 1; and b, c, and d are
 0. 96. Themethod of claim 87, wherein a, b, c, d, and e are members independentlyselected from 0 and
 1. 97. A G-CSF peptide conjugate formed by themethod of claim
 87. 98. A G-CSF peptide comprising one or more glycans,having a glycoconjugate molecule covalently attached to said peptide.99. The G-CSF peptide of claim 98, wherein said one or more glycans is amonoantennary glycan.
 100. The G-CSF peptide of claim 98, wherein saidone or more glycans is a biantennary glycan.
 101. The G-CSF peptide ofclaim 98, wherein said one or more glycans is a triantennary glycan.102. The G-CSF peptide of claim 98, wherein said one or more glycans isat least a triantennary glycan.
 103. The G-CSF peptide of claim 98,wherein said one or more glycans comprises at least two glycanscomprising a mixture of mono or multiantennary glycans.
 104. The G-CSFpeptide of claim 98, wherein said one or more glycans is selected froman N-linked glycan and an O-linked glycan.
 105. The G-CSF peptide ofclaim 98, wherein said one or more glycans is at least two glycansselected from an N-linked and an O-linked glycan.
 106. The G-CSF peptideof claim 98, wherein said peptide is expressed in a cell selected fromthe group consisting of a prokaryotic cell and a eukaryotic cell. 107.The G-CSF peptide of claim 106, wherein said eukaryotic cell is selectedfrom the group consisting of a mammalian cell, an insect cell and ayeast cell.
 108. A method of treating a mammal having a disease selectedfrom the group consisting of an infectious disease, acute myeloidleukemia, non-myeloid cancer, chronic or persistent neutropenia, saidmethod comprising administering to said mammal a G-CSF peptide havingone or more glycans having a glycoconjugate molecule attached to saidpeptide.
 109. The method of claim 108, wherein said infectious diseaseis selected from the group consisting of a bacterial and a viraldisease.
 110. The method of claim 108, wherein said glycoconjugatemolecule is poly(ethylene glycol).
 111. The method of claim 108, whereinsaid mammal is a human.